Anti-FLT-1 antibodies in the treatment of Duchenne muscular dystrophy

Abstract

To provide anti-FLT-1 antibodies in the treatment of Duchenne muscular dystrophy.SOLUTION: The present invention provides, among other things, anti-Flt-1 antibodies and methods for treating muscular dystrophy, in particular, Duchenne muscular dystrophy (DMD). In some embodiments, a method according to the present invention includes administering to an individual who is suffering from or susceptible to DMD an effective amount of an anti-Flt-1 antibody or antigen-binding protein thereof so as to reduce intensity, severity, or frequency of at least one symptom or feature of DMD, or to delay DMD onset.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 144,251, filed April 7, 2015, and U.S. Provisional Patent Application No. 62 / 307,645, filed March 14, 2016, the disclosures of each of which are incorporated herein by reference. [Background technology]

[0002] Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects approximately 1:3600 live birth males, with approximately 50,000 affected individuals worldwide. The disease is characterized by progressive muscle weakness, and affected children become wheelchair-bound by the age of 13. Affected individuals usually exhibit symptoms at age 3, and the average survival time for such individuals is between 25 and 30 years. Respiratory failure and cardiomyopathy due to diaphragmatic weakness are common causes of death.

[0003] DMD is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome and encodes the protein dystrophin. The dystrophin protein connects the contractile machinery of muscle fibers (the actin-myosin complex) to the surrounding extracellular matrix via the dystroglycan complex. Mutations in the dystrophin gene result in altered or absent dystrophin protein and abnormal muscle fiber membrane function. Both men and women can carry dystrophin gene mutations, but women are rarely affected by DMD.

[0004] One characteristic of DMD is ischemia in the affected tissue. Ischemia is a restriction or reduction in blood supply to a tissue or organ, resulting in a lack of oxygen and nutrients necessary for cellular metabolism. Ischemia is generally caused by constriction or blockage of blood vessels, resulting in damage to or dysfunction of the tissue or organ. Treatment of ischemia is performed to increase blood flow to the affected tissue or organ.

[0005] Currently, there is no cure for DMD. Although several therapeutic approaches have been explored, including gene therapy and corticosteroid administration, there remains a need for alternatives for DMD patients. Summary of the Invention [Means for solving the problem]

[0006] The present invention provides, inter alia, improved methods and compositions for treating muscular dystrophies, particularly Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy, based on anti-Flt-1 antibody therapy. As described in the Examples below, the present invention is based, in part, on the discovery that anti-Flt-1 antibodies, or antigen-binding fragments thereof, can block the binding of VEGF and other ligands to the Flt-1 receptor, thereby increasing the amount of VEGF and / or other ligands available to bind to the VEGF receptor. Increased VEGF availability promotes angiogenesis with increased blood flow to muscles to combat functional ischemia, resulting in improvement of the structural and functional characteristics of DMD. Indeed, as shown in these Examples, the inventors have demonstrated that administration of anti-Flt-1 antibodies improves the degree of muscle pathology (e.g., improved angiogenesis, reduced fibrosis, and reduced necrosis). Thus, the present invention provides safe and effective antibody-based therapies for the treatment of DMD.

[0007] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising one or more complementarity-determining regions (CDRs) selected from the group consisting of a variable light (VL) chain CDR1 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21; a VL CDR2 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24; a VL CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34; a variable heavy (VH) chain CDR1 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4; a VH CDR2 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14; and a VH CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0008] In some embodiments, the one or more CDRs include a VL CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34, and a VH CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18.

[0009] In another embodiment, the one or more CDRs comprise a VL CDR1 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21, a VL CDR2 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24, and a VL CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34. In a specific embodiment, the VL chain comprises VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NOs: 19, 22, and 25, respectively. In yet another embodiment, the VL chain comprises VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NOs: 20, 23, and 25, respectively. In another embodiment, the VL chain comprises a VL CDR1 and a VL CDR2 defined by the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:24, respectively, and a VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. In a specific embodiment, the VL chain comprises a VL CDR1, a VL CDR2, and a VL CDR3 defined by the amino acid sequences of SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:32, respectively.

[0010] In other embodiments, the one or more CDRs comprise a VH CDR1 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-4, a VH CDR2 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5-14, and a VH CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15-18. In a specific embodiment, the VH chain comprises a VH CDR1, a VH CDR2, and a VH CDR3 defined by the amino acid sequences of SEQ ID NOs: 1, 5, and 15, respectively. In another embodiment, the VH chain comprises a VH CDR1, a VH CDR2, and a VH CDR3 defined by the amino acid sequences of SEQ ID NOs: 2, 6, and 16, respectively. In yet another embodiment, the VH chain comprises a VH CDR1, a VH CDR2, and a VH CDR3 defined by the amino acid sequences of SEQ ID NOs: 2, 10, and 18, respectively. In another embodiment, the VH chain comprises a VH CDR1 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:17, respectively, and a VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14. In another embodiment, the VH chain comprises a VH CDR1 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:17, respectively. In yet another embodiment, the VH chain comprises a VH CDR1, a VH CDR2 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 12. In yet another embodiment, the VH chain comprises a VH CDR1, a VH CDR2 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO: 17, respectively. In a specific embodiment, the VH chain comprises a VH CDR1, a VH CDR2 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 12 and SEQ ID NO: 17, respectively.

[0011] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising: (i) a light chain variable (VL) region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 49 to 61, and / or (ii) a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 35 to 48. In a specific embodiment, the VL region comprises the amino acid sequence of SEQ ID NO: 60, and the VH region comprises the amino acid sequence of SEQ ID NO: 45.

[0012] In some embodiments, the antibody further comprises a heavy chain constant region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NO:87-89.

[0013] In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising (i) a light chain comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 75 to 86, and / or (ii) a heavy chain comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 62 to 74. In a specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 76, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 71.

[0014] In another embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of IgG, F(ab'), F(ab)2, Fab', Fab, ScFv, diabodies, triabodies, and tetrabodies. In one embodiment, the antibody or antigen-binding fragment thereof is an IgG. In another embodiment, the antibody or antigen-binding fragment thereof is an IgG1. In yet another embodiment, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In a specific embodiment, the antibody is a humanized monoclonal antibody. In yet another embodiment, the humanized monoclonal antibody contains a human Fc region. In some embodiments, the Fc region comprises one or more mutations that enhance the binding affinity between the Fc region and the FcRn receptor such that the in vivo half-life of the antibody is increased. In another embodiment, the Fc region comprises one or more mutations at positions corresponding to Leu234, Leu235, and / or Gly237 of human IgG1.

[0015] In one embodiment, the antibody or antigen-binding fragment thereof does not bind to VEGF R2 and / or VEGF R3.In another embodiment, the antibody or antigen-binding fragment thereof does not bind to mouse or monkey Flt-1.

[0016] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that recognizes a peptide or a fragment thereof comprising an amino acid sequence corresponding to positions 139-148, 139-153, 178-206, 199-204, and 128-138 of SEQ ID NO: 90. In one embodiment, the peptide consists of an amino acid sequence corresponding to positions 130-138, 141-148, 141-153, and 193-206 of SEQ ID NO: 90.

[0017] In another aspect, the present invention provides an isolated antibody or antigen-binding fragment thereof that competes with any anti-Flt-1 antibody or antigen-binding fragment thereof.

[0018] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-Flt-1 antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0019] In yet another aspect, the present invention provides polynucleotides encoding the CDRs, VL regions, VH regions, light chains, and / or heavy chains of the antibodies or antigen-binding fragments thereof of the present invention. In one embodiment, the present invention provides an expression vector comprising the polynucleotide. In yet another embodiment, the present invention provides a host cell comprising the polynucleotide or expression vector. In a specific embodiment, the present invention provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising culturing the host cell. In another embodiment, a hybridoma cell produces the antibody or antigen-binding fragment thereof.

[0020] In another aspect, the present invention provides a method for treating an Flt-1-mediated disease, disorder, or condition, comprising administering an anti-Flt-1 antibody or antigen-binding fragment thereof to a subject in need of treatment. In certain embodiments, the Flt-1-mediated disease, disorder, or condition is Duchenne muscular dystrophy, Becker muscular dystrophy, bronchopulmonary dysplasia, pre-eclampsia, or chronic kidney disease.

[0021] In another aspect, the present invention provides a method for treating Duchenne muscular dystrophy (DMD), the method comprising administering an effective amount of an anti-Flt-1 antibody or antigen-binding fragment thereof to a subject suffering from or susceptible to DMD, such that at least one symptom or feature of DMD is reduced in intensity, severity, or frequency, or the onset is delayed. In one embodiment, the method comprises administering to the subject one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent is selected from the group consisting of prednisone, deflazacort, follistatin, RNA modulation therapy, exon skipping therapy, and gene therapy.

[0022] In one embodiment, the antibody or antigen-binding fragment thereof is administered parenterally. In some embodiments, parenteral administration is selected from intravenous, intradermal, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, and / or transmucosal administration. In a specific embodiment, parenteral administration is intravenous administration. In yet another embodiment, parenteral administration is subcutaneous administration.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof is administered daily, twice weekly, weekly, or monthly. In a specific embodiment, the antibody or antigen-binding fragment thereof is administered twice weekly.

[0024] In another embodiment, an effective amount of an antibody or antigen-binding fragment thereof is administered at a dose of about 1 mg / kg to 50 mg / kg, hi specific embodiments, the dose is about 1 mg / kg, 3 mg / kg, or 10 mg / kg.

[0025] In one embodiment, administration of the antibody or antigen-binding fragment thereof results in reduced fibrosis and / or necrosis compared to a control. In another embodiment, administration of the antibody or antigen-binding fragment thereof results in improved vascularization of the subject's muscle compared to a control. In another embodiment, improved vascularization is reflected by increased blood flow to the muscle pathology, increased serum VEGF levels, decreased serum creatine kinase (CK) concentrations, increased CD31 score by IHC, and / or decreased serum sFlt-1 levels. In yet another embodiment, the antibody or antigen-binding fragment thereof results in improved muscle function compared to a control. In yet another embodiment, improved muscle function is reflected by improved muscle strength and / or resistance to fatigue.

[0026] In another aspect, the present invention provides a method of treating tissue fibrosis, comprising administering to a subject in need thereof an effective amount of an anti-Flt-1 antibody or antigen-binding fragment thereof.

[0027] The present teachings set forth herein will be more fully understood from the following description of various exemplary embodiments when read in conjunction with the accompanying drawings, in which it should be understood that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. In certain embodiments, for example, the following are provided: (Item 1) A variable light (VL) chain CDR1 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21; VL CDR2 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24; VL CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34; a variable heavy (VH) chain CDR1 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4; VH CDR2 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14; VH CDR3 defined by an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18; An antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising one or more complementarity-determining regions (CDRs) selected from the group consisting of: (Item 2) 2. The antibody or antigen-binding fragment thereof according to Item 1, wherein the one or more CDRs comprise the VL CDR3 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34, and the VH CDR3 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18. (Item 3) 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the one or more CDRs comprise the VL CDR1 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19 to 21, the VL CDR2 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 22 to 24, and the VL CDR3 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 25 to 34. (Item 4) 10. The antibody or antigen-binding fragment thereof according to any one of the preceding items, wherein the one or more CDRs comprise the VH CDR1 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1 to 4, the VH CDR2 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 5 to 14, and the VH CDR3 defined by the amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 to 18. (Item 5) The antibody or antigen-binding fragment thereof of any one of the preceding items, comprising a VL chain comprising the VL CDR1, VL CDR2 and VL CDR3 defined by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22 and SEQ ID NO: 25, respectively. (Item 6) 5. The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, comprising a VL chain comprising the VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 25, respectively. (Item 7) 5. The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, comprising a VL chain comprising the VL CDR1 and VL CDR2 defined by the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 24, respectively, and the VL CDR3 defined by the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. (Item 8) 5. The antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the VL chain comprises the VL CDR1, VL CDR2, and VL CDR3 defined by the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 24, and SEQ ID NO: 32, respectively. (Item 9) 10. The antibody or antigen-binding fragment thereof of any one of the preceding items, comprising a VH chain comprising the VH CDR1, VH CDR2 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:15, respectively. (Item 10) 9. The antibody or antigen-binding fragment thereof according to any one of items 1 to 8, comprising a VH chain comprising the VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 16, respectively. (Item 11) 9. The antibody or antigen-binding fragment thereof according to any one of items 1 to 8, comprising a VH chain comprising the VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 10, and SEQ ID NO: 18, respectively. (Item 12) 9. The antibody or antigen-binding fragment thereof according to any one of Aspects 1 to 8, comprising a VH chain comprising the VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 17, respectively, and the VH CDR2 defined by the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 13, or SEQ ID NO: 14. (Item 13) 9. The antibody or antigen-binding fragment thereof according to any one of Aspects 1 to 8, comprising a VH chain comprising the VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 3 and SEQ ID NO: 17, respectively, and the VH CDR2 defined by the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 12. (Item 14) 9. The antibody or antigen-binding fragment thereof according to any one of items 1 to 8, comprising a VH chain comprising the VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 9, and SEQ ID NO: 17, respectively. (Item 15) 9. The antibody or antigen-binding fragment thereof according to any one of items 1 to 8, comprising a VH chain comprising the VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 17, respectively. (Item 16) An antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising: (i) a light chain variable (VL) region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 49 to 61; and / or (ii) a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 35 to 48. (Item 17) 17. The antibody or antigen-binding fragment thereof according to item 16, wherein the VL region comprises the amino acid sequence of SEQ ID NO: 60 and the VH region comprises the amino acid sequence of SEQ ID NO: 45. (Item 18) The antibody of any one of the preceding items, wherein the antibody further comprises a heavy chain constant region comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 87 to 89. (Item 19) An antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising: (i) a light chain comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 75 to 86; and / or (ii) a heavy chain comprising an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 62 to 74. (Item 20) 20. The antibody or antigen-binding fragment thereof of item 19, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 76 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 71. (Item 21) 2. The antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of IgG, F(ab')2, F(ab)2, Fab', Fab, scFv, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. (Item 22) 22. The antibody or antigen-binding fragment thereof of item 21, wherein the antibody or antigen-binding fragment thereof is an IgG. (Item 23) 23. The antibody or antigen-binding fragment thereof of item 22, wherein the antibody or antigen-binding fragment thereof is an IgG1. (Item 24) 8. The antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody. (Item 25) 2. The antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the antibody is a humanized monoclonal antibody. (Item 26) 26. The antibody or antigen-binding fragment thereof of item 25, wherein the humanized monoclonal antibody contains a human Fc region. (Item 27) 27. The antibody or antigen-binding fragment thereof of item 26, wherein the Fc region comprises one or more mutations that enhance the binding affinity between the Fc region and the FcRn receptor such that the antibody has a longer in vivo half-life. (Item 28) Item 10. The antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the Fc region comprises one or more mutations at positions corresponding to Leu234, Leu235 and / or Gly237 of human IgG1. (Item 29) Item 10. The antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the antibody or antigen-binding fragment thereof does not bind to VEGF R2 and / or VEGF R3. (Item 30) The antibody or antigen-binding fragment thereof of any one of the preceding items, wherein the antibody or antigen-binding fragment thereof does not bind to mouse or monkey Flt-1. (Item 31) An isolated antibody or antigen-binding fragment thereof that recognizes a peptide or a fragment thereof comprising an amino acid sequence corresponding to positions 139 to 148, 139 to 153, 178 to 206, 199 to 204, and 128 to 138 of SEQ ID NO: 90. (Item 32) 32. The isolated antibody or antigen-binding fragment thereof according to Item 31, wherein the peptide consists of the amino acid sequences corresponding to positions 130 to 138, positions 141 to 148, positions 141 to 153, and positions 193 to 206 of SEQ ID NO: 90. (Item 33) An isolated antibody or antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment thereof of any one of the preceding items. (Item 34) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of the preceding items and a pharmaceutically acceptable carrier. (Item 35) A polynucleotide encoding the CDR, VL region, VH region, light chain and / or heavy chain of the antibody or antigen-binding fragment thereof according to any one of items 1 to 33. (Item 36) 36. An expression vector comprising the polynucleotide of Item 35. (Item 37) A host cell comprising the polynucleotide of Item 35 and the expression vector of Item 36. (Item 38) 38. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising culturing the host cell of Item 37. (Item 39) A hybridoma cell that produces the antibody or antigen-binding fragment thereof according to any one of items 1 to 33. (Item 40) Item 41. A method for treating an Flt-1-mediated disease, disorder, or condition, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof described in any one of Items 1 to 33. Item 42. The method of item 40, wherein the Flt-1-mediated disease, disorder, or condition is Duchenne muscular dystrophy, Becker muscular dystrophy, preeclampsia, or chronic kidney disease. 1. A method of treating Duchenne muscular dystrophy (DMD), said method comprising: 36. The method of claim 35, comprising administering to a subject suffering from or susceptible to DMD an effective amount of the antibody or antigen-binding fragment thereof described in any one of items 1 to 35, such that at least one symptom or feature of DMD is reduced in intensity, severity, or frequency, or the onset of DMD is delayed. (Item 43) 43. The method of claim 42, wherein the method comprises administering to the subject one or more additional therapeutic agents. (Item 44) 44. The method of item 43, wherein the one or more additional therapeutic agents are selected from the group consisting of prednisone, deflazacort, follistatin, RNA modulatory therapy, exon skipping therapy, and gene therapy. (Item 45) 45. The method of any one of items 42 to 44, wherein the antibody or antigen-binding fragment thereof is administered parenterally. (Item 46) 46. ​​The method of item 45, wherein the parenteral administration is selected from intravenous, intradermal, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous and / or transmucosal administration. (Item 47) 47. The method of claim 46, wherein the parenteral administration is intravenous administration. (Item 48) 47. The method of claim 46, wherein the parenteral administration is subcutaneous administration. (Item 49) 49. The method of any one of items 42 to 48, wherein the antibody or antigen-binding fragment thereof is administered daily, twice weekly, weekly, or monthly. (Item 50) 50. The method of item 49, wherein the antibody or antigen-binding fragment thereof is administered twice weekly. (Item 51) 49. The method according to any one of items 42 to 48, wherein the effective amount of the antibody or antigen-binding fragment thereof is administered at a dose of about 1 mg / kg to 50 mg / kg. (Item 52) 52. The method of claim 51, wherein the dosage is about 1 mg / kg. (Item 53) 52. The method of claim 51, wherein the dosage is about 3 mg / kg. (Item 54) 52. The method of claim 51, wherein the dosage is about 10 mg / kg. (Item 55) 55. The method of any one of items 42 to 54, wherein the administration of the antibody or antigen-binding fragment thereof results in reduced fibrosis and / or necrosis compared to a control. (Item 56) 55. The method of any one of items 42 to 54, wherein the administration of the antibody or antigen-binding fragment thereof results in improved vascularization of muscle in the subject compared to a control. (Item 57) 57. The method of item 56, wherein the improved angiogenesis is reflected by increased blood flow to the muscle pathology, increased serum VEGF levels, decreased serum creatine kinase (CK) concentrations, increased CD31 score by IHC, and / or decreased serum sFlt-1 levels. (Item 58) 55. The method of any one of items 42 to 54, wherein the administration of the antibody or antigen-binding fragment thereof results in improved muscle function compared to a control. (Item 59) 59. The method of item 58, wherein the improved muscle function is reflected in improved muscle strength and / or resistance to fatigue. (Item 60) 34. A method for treating tissue fibrosis, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 33. [Brief explanation of the drawings]

[0028] [Figure 1A]1 shows exemplary results demonstrating clearance of anti-Flt-1 antibody 13B4 after intravenous administration of the antibody to mice at a dose of 10 mg / kg. [Figure 1B] 1 shows exemplary results demonstrating clearance of anti-Flt-1 antibody 10G12 after intravenous administration of the antibody to mice at a dose of 10 mg / kg. [Figure 2A] 1 shows exemplary results showing peak serum concentrations of anti-Flt-1 antibodies. [Figure 2B] 1 shows exemplary results showing the lowest serum concentrations of anti-Flt-1 antibodies. [Figure 3] 1 shows exemplary results demonstrating the reduction of free soluble Flt-1 (sFlt-1) concentrations in serum after administration of anti-Flt-1 antibodies (13B4 or 10G12), an isotype control antibody, a commercial antibody (Angio), or vehicle alone to mdx mice. [Figure 4] 1 shows exemplary results demonstrating an increase in serum levels of VEGF following administration of anti-Flt-1 antibodies (13B4 or 10G12), an isotype control antibody, a commercial antibody (Angio), or vehicle alone to mdx mice. [Figure 5] Figures 5A-5D show exemplary results of CD31 staining of tissue sections obtained from the diaphragm muscle of mdx mice administered anti-Flt-1 antibodies (13B4 or 10G12), an isotype control antibody, or a commercially available antibody (Angio). [Figure 6A] Exemplary results of quantification of CD31-positive areas as a percentage of all stained areas of tissue sections obtained from diaphragm muscles are shown. [Figure 6B] Figure 6B shows exemplary results of quantification of CD31-positive areas as a percentage of all colored areas in tissue sections obtained from the tibialis anterior (TA) muscle of mdx mice administered anti-Flt-1 antibody. [Figure 7] 1 shows exemplary results demonstrating binding of anti-Flt-1 antibodies to recombinant sFlt-1 by ELISA assay. [Figure 8]1 shows exemplary results demonstrating inhibition of sFlt-1 binding to VEGF by anti-Flt-1 antibodies in a competitive ELISA assay. [Figure 9] 1 shows exemplary results demonstrating the rescue of VEGF R2 phosphorylation by anti-Flt-1 antibody. Human vein endothelial cells (HUVECs) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody, and the level of VEGF R2 phosphorylation was determined. % rescue indicates the level of VEGF R2 phosphorylation compared to the level of VEGF R2 phosphorylation when HUVECs were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody). [Figure 10] 1 shows exemplary results demonstrating the inhibition of soluble Flt-1 binding to VEGF by anti-Flt-1 antibodies in a competitive ELISA assay. [Figure 11] 1 shows exemplary results showing the disappearance of anti-Flt-1 antibodies from serum 672 hours after intravenous administration of anti-Flt-1 antibodies to mice at a dose of 10 mg / kg. [Figure 12] Figures 12A-12C show exemplary results of CD31 staining of tissue sections from the diaphragm muscle, and Figures 12D-12F show exemplary results of CD31 staining of tissue sections obtained from the tibialis anterior muscle. [Figure 13A] 1 shows exemplary biodistribution of anti-Flt-1 antibodies 27H9, 13B4, and 21B3 in mouse diaphragm, tibia, and gastrocnemius muscles over a time course of 256 hours after antibody administration. [Figure 13B] 1 shows exemplary biodistribution of anti-Flt-1 antibodies 27H9, 13B4, and 21B3 in mouse diaphragm, tibia, and gastrocnemius muscles over a time course of 256 hours after antibody administration. [Figure 13C] 1 shows exemplary biodistribution of anti-Flt-1 antibodies 27H9, 13B4, and 21B3 in mouse diaphragm, tibia, and gastrocnemius muscles over a time course of 256 hours after antibody administration. [Figure 14A] 1 shows exemplary results depicting the highest anti-Flt-1 antibody 21B3 concentrations at maximum exposure. [Figure 14B]1 shows exemplary results showing the lowest anti-Flt-1 antibody 21B3 concentrations. [Figure 15] 1 shows exemplary results depicting free sFlt-1 following administration of anti-Flt-1 antibody 21B3 to mdx mice. [Figure 16] 1 shows exemplary results showing VEGF levels after administration of anti-Flt-1 antibody 21B3 to mdx mice. [Figure 17] 17A-17E show exemplary results of CD31 staining of tissue sections obtained from diaphragm muscles of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody. [Figure 18] 1 shows exemplary results of quantification of normalized CD31 positive percentages in tissue sections obtained from diaphragm muscles of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody. [Figure 19] 19A-19E show exemplary results of CD31 staining of tissue sections obtained from the tibialis anterior muscle of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody. [Figure 20] 1 shows exemplary results of quantification of normalized CD31 positive percentages in tissue sections obtained from the tibialis anterior muscles of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody. [Figure 21A] 1 shows exemplary results of reverse phase liquid chromatography / mass spectrometry (RP-LC / MS) analysis to determine the molecular weight of deglycosylated 21B3 antibody. [Figure 21B] 1 shows exemplary results of an analysis of the glycosylation pattern of the heavy chain. [Figure 22A] 1 shows exemplary results demonstrating the rescue of VEGF R2 phosphorylation by anti-Flt-1 antibody. Human vein endothelial cells (HUVECs) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody, and the level of VEGF R2 phosphorylation was determined. % rescue indicates the level of VEGF R2 phosphorylation compared to the level of VEGF R2 phosphorylation when HUVECs were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody). [Figure 22B]1 shows exemplary results demonstrating the rescue of VEGF R2 phosphorylation by anti-Flt-1 antibody. Human vein endothelial cells (HUVECs) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody, and the level of VEGF R2 phosphorylation was determined. % rescue indicates the level of VEGF R2 phosphorylation compared to the level of VEGF R2 phosphorylation when HUVECs were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody). [Figure 23] 1 shows exemplary results demonstrating binding of anti-Flt-1 antibodies to recombinant sFlt-1 by ELISA assay. [Figure 24] 1 shows exemplary results showing serum concentrations of free anti-Flt-1 antibody 21B3 and an isotype control antibody in mdx mice. [Figure 25] 1 shows exemplary results depicting serum concentrations of free sFlt-1 in mdx mice treated with either anti-Flt-1 antibody 21B3 or an isotype control antibody. [Figure 26] 1 shows exemplary results depicting serum levels of VEGF in mdx mice treated with either the anti-Flt-1 antibody 21B3 or an isotype control antibody. [Figure 27] 27A-27H show exemplary results of CD31 staining of tissue sections obtained from diaphragm muscle of mdx mice administered anti-Flt-1 antibody 21B3 or solvent control for 6 weeks (27A-27D) or 12 weeks (27E-27H). [Figure 28] 28A-28H show exemplary results of CD31 staining of tissue sections obtained from the gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or solvent control for 6 weeks (28A-28D) or 12 weeks (28E-28H). [Figure 29] 29A-29H show exemplary results of CD31 staining of tissue sections obtained from the tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (29A-29D) or 12 weeks (29E-29H). [Figure 30A]1 shows exemplary results of quantification of % positive CD31 staining in tissue sections from diaphragm, gastrocnemius, and tibialis muscles of mdx mice treated with anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 or 12 weeks. [Figure 30B] 1 shows exemplary results of quantification of % positive CD31 staining in tissue sections from diaphragm, gastrocnemius, and tibialis muscles of mdx mice treated with anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 or 12 weeks. [Figure 30C] 1 shows exemplary results of quantification of % positive CD31 staining in tissue sections from diaphragm, gastrocnemius, and tibialis muscles of mdx mice treated with anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 or 12 weeks. [Figure 31] Figures 31A-31H show exemplary results of type I collagen immunohistochemical staining of tissue sections obtained from the diaphragm muscle of mdx mice administered anti-Flt-1 antibody 21B3 or solvent control for 6 weeks (31A-31D) or 12 weeks (31E-31H). [Figure 32] Figures 32A-32H show exemplary results of type I collagen immunohistochemical staining of tissue sections obtained from the gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (32A-32D) or 12 weeks (32E-32H). [Figure 33] Figures 33A-33H show exemplary results of type I collagen immunohistochemical staining of tissue sections obtained from the tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control for 6 weeks (33A-33D) or 12 weeks (33E-33H). [Figure 34A] 1 shows exemplary results of quantification of percent positive type I collagen staining in tissue sections from diaphragm, gastrocnemius, and tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 or 12 weeks. [Figure 34B] 1 shows exemplary results of quantification of percent positive type I collagen staining in tissue sections from diaphragm, gastrocnemius, and tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 or 12 weeks. [Figure 34C]1 shows exemplary results of quantification of percent positive type I collagen staining in tissue sections from diaphragm, gastrocnemius, and tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or vehicle control antibody for 6 or 12 weeks. [Figure 35] 35A and 35B show exemplary results of quantification of % necrosis in gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or solvent control antibody for 6 or 12 weeks. [Figure 36-1] Figure 36 shows exemplary results depicting a differential heat map comparing the hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and an anti-Flt-1 antibody (21B3). Gray: no deuterium protection; Blue: deuterium protection upon Fab binding. [Figure 36-2] Figure 36 shows exemplary results depicting a differential heat map comparing the hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and an anti-Flt-1 antibody (21B3). Gray: no deuterium protection; Blue: deuterium protection upon Fab binding. [Figure 36-3] Figure 36 shows exemplary results depicting a differential heat map comparing the hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and an anti-Flt-1 antibody (21B3). Gray: no deuterium protection; Blue: deuterium protection upon Fab binding. [Figure 37-1] Figure 37 shows exemplary results depicting a differential heat map comparing the hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and an anti-Flt-1 antibody (21C6). Gray: no deuterium protection; Blue: deuterium protection upon Fab binding. [Figure 37-2] Figure 37 shows exemplary results depicting a differential heat map comparing the hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and an anti-Flt-1 antibody (21C6). Gray: no deuterium protection; Blue: deuterium protection upon Fab binding. [Figure 37-3] Figure 37 shows exemplary results depicting a differential heat map comparing the hydrogen / deuterium exchange of human sFlt-1 alone with that of a mixture of human sFlt-1 and an anti-Flt-1 antibody (21C6). Gray: no deuterium protection; Blue: deuterium protection upon Fab binding. [Figure 38A] 1 shows exemplary results depicting MS / MS spectra for identified peptides containing amino acid residues from the epitope region. [Figure 38B] 1 shows exemplary results depicting MS / MS spectra for identified peptides containing amino acid residues from the epitope region. [Figure 38C] 1 shows exemplary results depicting MS / MS spectra for identified peptides containing amino acid residues from the epitope region. [Figure 38D] 1 shows exemplary results depicting MS / MS spectra for identified peptides containing amino acid residues from the epitope region. [Figure 38E] 1 shows exemplary results depicting MS / MS spectra for identified peptides containing amino acid residues from the epitope region. DETAILED DESCRIPTION OF THE INVENTION

[0029] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0030] Affinity: As is well known in the art, "affinity" refers to the degree of tightness with which a particular ligand binds to its partner. In some embodiments, the ligand or partner is Flt-1. In some embodiments, the ligand or partner is soluble Flt-1. In some embodiments, the ligand or partner is recombinant Flt-1. In certain embodiments, the ligand or partner is human sFlt-1. In certain embodiments, the ligand or partner is recombinant sFlt-1. In other embodiments, the ligand or partner is an anti-Flt-1 antibody. Affinity can be measured in a variety of ways. In some embodiments, affinity is measured in a quantitative assay. In some such embodiments, the concentration of the binding partner can be fixed above the concentration of the ligand to mimic physiological conditions. Alternatively, or additionally, in some embodiments, the binding partner concentration and / or the ligand concentration can be varied. In some such embodiments, affinity can be compared to a reference under similar conditions (e.g., concentration).

[0031] Affinity maturation (or affinity matured antibody): As used herein, the term "affinity matured" or "affinity matured antibody" refers to an antibody that has one or more alterations in one or more of its CDRs that result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have the alteration(s). In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be produced by any of a variety of techniques well known in the art. Marks et al., BioTechnology 10:779-783 (1992), V H and V LAffinity maturation by domain shuffling has been described. Random mutagenesis of CDR and / or framework residues has been described in Barbas et al., Proc. Nat. Acad. Sci. USA 91:3809-3813 (1994), Schier et al., Gene 169:147-155 (1995), Yelton et al., J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0032] Amelioration: As used herein, the term "amelioration" refers to the prevention, alleviation, or alleviation of a condition, or improvement of a condition in a subject. Amelioration includes, but does not require, complete reversal or complete prevention of a disease condition.

[0033] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0034] Antibody: As used herein, the term "antibody" refers to any immunoglobulin, natural or wholly or partially synthetically produced. The term also encompasses all derivatives thereof that maintain specific binding ability. The term also covers any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources or can be partially or wholly synthetically produced. An antibody can be monoclonal or polyclonal. An antibody can also be a member of any immunoglobulin isotype, including any of the human isotypes: IgG, IgM, IgA, IgD, and IgE. In certain embodiments, an antibody can be a member of the IgG immunoglobulin class (e.g., IgG1, IgG2, IgG3, etc.). In some embodiments, the antibody can be a human antibody. In some embodiments, the antibody can be a humanized antibody.

[0035] As is well known to those skilled in the art, naturally occurring antibodies typically consist of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy and light chain contains a variable region (HCVR, VH or V, respectively). H and LCVR, VL or V L The heavy chain constant region consists of a C H 1. C H 2 and C H 3 domains (and optionally C for IgM and IgE) H The light chain constant region contains one domain, C L V H and V L The region further contains regions of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). H and V LEach antibody consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The binding regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0036] Antigen-binding portion: As used herein, the term "antigen-binding portion" or "antigen-binding fragment" refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., Flt-1). Examples of antigen-binding portions include: (i) V H , V L , C H 1 and C L (ii) a Fab fragment, which is a monovalent fragment consisting of two Fab fragments linked by disulfide bridges in the hinge region; (iii) a V H and C H (iv) a single-arm V of an antibody; H and V L Examples of such fragments include Fv fragments consisting of a single variable domain, (v) dAb fragments containing a single variable domain (Ward et al., (1989) Nature 341:544-546), (vi) isolated complementarity-determining regions (CDRs), (vii) Fab' fragments, which are essentially Fab fragments with a portion of the hinge region, and (viii) nanobodies, which are heavy chain variable regions containing a single variable domain and two constant regions. Furthermore, the two domains of the Fv fragment, V, L and V H Although the V and V proteins are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods, and in this chain, V L and V HThe domains pair to form a monovalent molecule (known as a single-chain Fv (scFv) (see, e.g., Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Antigen-binding fragments of antibodies can optionally comprise single-chain antibody fragments. Alternatively, or additionally, antigen-binding fragments of antibodies can comprise multiple chains linked, for example, by disulfide bonds. Antigen-binding fragments of antibodies can optionally comprise multimolecular complexes. Functional antibody fragments typically comprise at least about 50 amino acids, and more usually at least about 200 amino acids.

[0037] In some embodiments, an antibody fragment comprises sufficient sequence of a parent antibody that it binds to the same antigen as the parent antibody, hi some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen.

[0038] Those skilled in the art will understand that the term "antibody fragment" does not imply or limit any particular state of production. Antibody fragments can be prepared using any suitable method, including, but not limited to, cleavage of intact antibodies, chemical synthesis, and recombinant production. Fragments are screened for uses in the same manner as intact antibodies.

[0039] Approximately or about: As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value, unless otherwise specified or apparent from the context (except where such number exceeds 100% of the possible values).

[0040] Associated with: As the term is used herein, two events or entities are "associated" with one another if the presence, level, and / or form of one correlates with the other. For example, if the presence, level, and / or form of a particular entity correlates with the incidence of and / or susceptibility to a particular disease, disorder, or condition (e.g., across a relevant population), that particular entity (e.g., a polypeptide) is considered associated with that disease, disorder, or condition. In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly, such that they are in and remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bonded to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently bonded to one another, but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0041] Carrier or diluent: As used herein, the terms "carrier" and "diluent" refer to a pharmaceutically acceptable (e.g., safe and non-toxic for human administration) carrier or diluent useful for preparing a pharmaceutical formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[0042] CDR: As used herein, refers to a complementarity-determining region within an antibody variable region. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. A "set of CDRs" or "CDR set" refers to a group of three or six CDRs that occur in either a single variable region capable of binding to an antigen, or in the CDRs of cognate heavy and light chain variable regions capable of binding to an antigen. Certain systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.). Those skilled in the art recognize the differences between and among these systems and are able to understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0043] Chimeric antibody: As used herein, a chimeric antibody is an antibody whose amino acid sequence is identical to that found in a first species. H and V L In many embodiments, a chimeric antibody refers to an antibody that comprises a murine V antibody that binds to a human constant region, as well as a constant region sequence found in a second species that is different from the first species. H and V L In some embodiments, a human V region binds to a non-human constant region (e.g., a mouse constant region). H and V L Antibodies having such regions are called "reverse chimeric antibodies."

[0044] Dosage Form: As used herein, the terms "dosage form" and "unit dosage form" refer to a physically discrete unit of a therapeutic protein (e.g., an antibody) for a patient to receive treatment. Each unit contains a predetermined amount of active material calculated to produce a desired therapeutic effect. It should be understood, however, that the total amount of the composition to be administered will be determined by the attending physician within the scope of sound medical judgment.

[0045] Dysfunction: As used herein, the term "dysfunction" refers to an abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease in activity associated with such molecule. Dysfunction of a molecule can be caused by a defect associated with the molecule itself or with other molecules that directly or indirectly interact with or regulate the molecule.

[0046] Epitope: As used herein, includes any portion that is specifically recognized by an immunoglobulin (e.g., antibody, antibody fragment thereof, receptor) binding component. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional structure. In some embodiments, such chemical atoms or groups are physically close to each other via gaps when the antigen adopts such a structure. In some embodiments, at least some such chemical atoms and groups are physically separated from each other when the antigen adopts another structure (e.g., linearized).

[0047] Fc region: As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each of which comprises the constant region of an antibody, excluding the first constant region immunoglobulin domain. In some embodiments, an "Fc region" comprises two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include part or all of the flexible hinge N-terminal to these regions. In IgG, the "Fc polypeptide" comprises immunoglobulin domains C gamma 2 (Cγ2) and C gamma 3 (Cγ3), as well as the lower part of the hinge between C gamma 1 (Cγ1) and Cγ2. Although the boundaries of an Fc polypeptide can vary, the human IgG heavy chain Fc polypeptide is usually defined to include residues starting at T223, or C226, or P230 relative to the carboxyl terminus, numbering according to the EU index of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). In IgA, the Fc polypeptide includes immunoglobulin domains C alpha 2 (Cα2) and C alpha 3 (Cα3), as well as the lower portion of the hinge between C alpha 1 (Cα1) and Cα2. The Fc region can be synthetic, recombinant, or produced from natural sources, such as IVIG.

[0048] Framework or framework region: As used herein, this refers to the sequence of a variable region excluding the CDRs. Because CDR sequences can be determined differently, framework sequences are also subject to correspondingly different interpretations. The six CDRs divide the framework regions on the heavy and light chains into four subregions (FR1, FR2, FR3, and FR4) on each chain. Among them, CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Framework regions referred to by others without specifying a particular subregion as FR1, FR2, FR3, or FR4 represent the combined FRs in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions. For example, FR1 represents the first framework region closest to the amino terminus of the variable region and 5'-most relative to CDR1, and FR represents two or more subregions that make up the framework region.

[0049] Half-life: As used herein, the term "half-life" is the time required for a quantity, such as a protein concentration or activity, to fall to half of its value measured at the beginning of the period.

[0050] High affinity: As used herein, the term "high affinity" when referring to an IgG type antibody refers to an antibody that binds to an Flt-1 domain with a binding affinity of 10 -8 M or less, preferably 10 -9 M or less, and even more preferably 10 -10 K below M D However, "high affinity" binding may differ for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a -7 M or less, preferably 10 -8 M or less, and even more preferably 10 -9 K below M D It refers to an antibody having the following structure:

[0051] Human antibody: As used herein, is intended to include antibodies having variable and constant regions generated from (or assembled from) human immunoglobulin sequences. In some embodiments, an antibody (or antibody component) can be considered "human" even if its amino acid sequence, for example in one or more CDRs, particularly CDR3, includes residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., sequence variations that may be introduced (ab initio) by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo).

[0052] Human monoclonal antibody: As used herein, is intended to refer to antibodies displaying a single binding specificity which have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas, which comprise B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) whose genome comprises human heavy chain and light chain transgenes fused to an immortalized cell.

[0053] Humanized: As is well known in the art, the term "humanized" refers to a process in which the amino acid sequence is modified from a V antibody from a reference antibody originating in a non-human species (e.g., mouse, llama). H and V LThe term "humanized" is generally used to refer to antibodies (or antibody components) that contain sequences of the target and / or target regions, but also contain modifications in those sequences relative to the reference antibody intended to make them more "human-like," i.e., more similar to human germline variable sequences. In some embodiments, a "humanized" antibody (or antibody component) is one that immunospecifically binds to an antigen of interest and has framework (FR) regions that have substantially the same amino acid sequences as those of a human antibody, and complementarity-determining regions (CDRs) that have substantially the same amino acid sequences as those of a non-human antibody (e.g., mouse, llama). A humanized antibody contains substantially all of at least one, and usually two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor immunoglobulin), and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also contains at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody comprises both a light chain and at least a heavy chain variable domain. H 1. Hinge, C H 2. C H 3, and optionally C H In some embodiments, the humanized antibody may also comprise a humanized V4 region. L In some embodiments, the humanized antibody comprises only a humanized V H In some embodiments, the humanized antibody comprises only a humanized V H and V L Includes the area.

[0054] Hypertrophy: As used herein, the term "hypertrophy" refers to an increase in the volume of an organ or tissue resulting from enlargement of the organ's or tissue's constituent cells.

[0055] Improve, increase, or decrease: As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to a value relative to a baseline measurement, e.g., a measurement in the same individual before the initiation of a treatment described herein, or a measurement in a control individual(s) not receiving a treatment described herein. A "control individual" is an individual suffering from the same form of disease as the individual being treated, and who is approximately the same age as the individual being treated (so as to ensure a comparison of the stage of disease in the individual being treated and the control individual(s)).

[0056] Inhibition: As used herein, the terms "inhibition," "inhibit," and "inhibiting" refer to processes and methods that decrease or reduce the activity and / or expression of a protein or gene of interest. Typically, inhibiting a protein or gene means a reduction in expression or associated activity of the protein or gene by at least 10% or more, e.g., 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or more, or a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more reduction in expression or associated activity, as measured by one or more methods described herein or recognized by those of skill in the art.

[0057] In Vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multicellular organism.

[0058] In Vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0059] Isolated antibody: As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to Flt-1). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0060] K a : as used herein refers to the binding rate of a particular antibody-antigen interaction, and as used herein "K d The term "K" is intended to refer to the off-rate of a particular antibody-antigen interaction. D The term "dissociation constant" is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to K (i.e., Kd / K) and expressed as a molar concentration (M). D The K value can be determined using methods well established in the art. D A preferred method for determining is by using surface plasmon resonance, preferably using a biosensor system (eg, a BIAcore® system).

[0061] Light chain reshuffling: As used herein, the term "light chain reshuffling" is intended to refer to an affinity maturation step in which the heavy chain sequence is held constant and a library of light chain sequences is generated. The light chain library is screened against the heavy chain to identify antibodies with improved binding affinity. The improved binding affinity can be in the nanomolar or picomolar range.

[0062] Monoclonal antibody: As used herein, the term "monoclonal antibody" is intended to refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0063] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to a material that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0064] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids joined via peptide bonds. While the term is used to refer to an amino acid chain of any length, those skilled in the art will understand that the term is not limited to long chains, but can also refer to a minimal chain comprising two amino acids joined via a peptide bond. As known to those skilled in the art, polypeptides can be processed and / or modified.

[0065] Prevent: As used herein, the terms "prevent" or "prevention," when used in reference to the occurrence of a disease, disorder, and / or condition, refer to reducing the risk of developing the disease, disorder, and / or condition. See definition of "risk."

[0066] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as individual units. The terms "polypeptide" and "protein" can be used interchangeably if a single polypeptide is a separately functional unit and does not require permanent or temporary physical association with other polypeptides to form the separate functional unit. When the separate functional unit consists of two or more polypeptides that are physically associated with each other, the term "protein" refers to the multiple polypeptides that are physically coupled and function together as a separate unit.

[0067] Risk: As understood from the context, "risk" of a disease, disorder, and / or condition includes the likelihood that a particular individual will develop the disease, disorder, and / or condition (e.g., DMD). In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90% up to 100%. In some embodiments, risk is expressed relative to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of the disease, disorder, condition, and / or event (e.g., DMD). In some embodiments, the reference sample or group of reference samples is obtained from individuals comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0068] Selective binding: As used herein, "selective binding," "selectively binds," "specific binding," or "specifically binds," with respect to a binding moiety and a target, refers to the preferential binding of the binding moiety to the target rather than to non-target entities. Some non-specific binding may occur between the binding moiety and the non-target. In some embodiments, a binding moiety selectively binds to a target if the binding between the binding moiety and the target is more than 2-fold, more than 5-fold, more than 10-fold, or more than 100-fold greater than the binding between the binding moiety and the non-target. In some embodiments, a binding affinity of about 10 -5 Less than M, about 10 -6 Less than M, about 10 -7 Less than M, about 10 -8 Less than M or about 10 -9 If M is less than M, the binding moiety selectively binds to the target.

[0069] Striated muscle: As used herein, the term "striated muscle" refers to multinucleated muscle tissue under voluntary control with a regular arrangement of sarcomeres, the intracellular contractile units of muscle tissue that give it a striated appearance under a microscope. Typically, striated muscle can be cardiac, skeletal, and branchial arch muscles.

[0070] Smooth muscle: As used herein, the term "smooth muscle" refers to involuntarily controlled, non-striated muscle, including unicomponent and multicomponent muscles.

[0071] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes prenatal and postnatal states. In many embodiments, a subject is a human. A subject can be a patient, which refers to a person who visits a healthcare provider for diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be suffering from or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0072] Substantially: As used herein, the term "substantially" refers to the qualitative condition of indicating the total or near-total extent or degree of a characteristic or property of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion, or rarely, if ever, achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0073] Substantially homologous: The phrase "substantially homologous" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues at corresponding positions. Homologous residues can be identical residues. Alternatively, homologous residues can be non-identical residues with appropriately similar structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are usually classified as "hydrophobic" or "hydrophilic" amino acids and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type can often be considered a "homologous" substitution.

[0074] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., "Basic Local Alignment Search Tool," J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., "Methods in Enzymology," Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., "Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins," Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned programs usually provide an indication of the degree of homology. In some embodiments, two sequences are considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over the relevant stretch of residues, which in some embodiments is the complete sequence.In some embodiments, the relevant sequence is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0075] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercially available computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul, et al., Methods in Enzymology; Altschul, et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above-mentioned programs usually provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues, which in some embodiments is the complete sequence.In some embodiments, the relevant sequence is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0076] Surface plasmon resonance: as used herein refers to an optical phenomenon that allows for the analysis of specific binding interactions in real time by detecting changes in protein concentration within a biosensor matrix, such as by using a Biacore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U., et al. See, e.g., Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26, Jonsson, U., et al. (1991) Biotechniques 11:620-627, Johnson, B., et al. (1995) J. Mol. Recognit. 8:125-131, and Johnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0077] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition, eg, DMD.

[0078] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, condition, or event (e.g., DMD) may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, condition, and / or event; or (5) having undergone, planned to undergo, or required a transplant. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder and / or condition does not develop the disease, disorder and / or condition.

[0079] Target tissue: As used herein, the term "target tissue" refers to any tissue afflicted with a disease to be treated, such as DMD. In some embodiments, the target tissue includes tissue that displays pathology, symptoms, or characteristics associated with the disease, including, but not limited to, muscle wasting, skeletal deformity, cardiomyopathy, muscle ischemia, cognitive impairment, and impaired respiratory function. In some embodiments, the target tissue is smooth muscle, striated muscle, or cardiac muscle.

[0080] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of a symptom(s) of the disease, disorder, and / or condition. Those skilled in the art will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0081] Treatment: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition, such as DMD. Treatment may be applied to subjects who do not show signs of the disease and / or who show only early signs of the disease, with the intent of reducing the risk of developing pathology associated with the disease.

[0082] Detailed Description The present invention provides, inter alia, methods and compositions for treating muscular dystrophy, including Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy, based on the use of an anti-Flt-1 antibody or antigen-binding fragment thereof as a therapeutic agent for the treatment of muscular dystrophy. In some embodiments, the present invention provides a method of treating DMD, comprising administering a therapeutically effective amount of an anti-Flt-1 antibody or antigen-binding fragment thereof to an individual suffering from or susceptible to DMD, such that at least one symptom or characteristic of DMD is reduced in intensity, severity, or frequency, or its onset is delayed.

[0083] Various aspects of the present invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section may apply to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated.

[0084] Duchenne muscular dystrophy (DMD) DMD is a disease characterized by progressive muscle wasting and loss of muscle-related function throughout the body. The present invention contemplates providing methods and compositions for slowing, delaying, or preventing muscle wasting, regenerating muscle, and reversing, eliminating, delaying, preventing, or minimizing fibrosis, inflammation, and other symptoms or characteristics associated with DMD and other muscular dystrophies in various muscle tissues.

[0085] muscle tissue There are two major types of muscle tissue in animals: striated and smooth. As used herein, the term "striated muscle" refers to muscle tissue containing continuous sarcomeres. Striated muscles are under voluntary control and tend to be attached to the skeleton. Striated muscles enable voluntary movement of the body and comprise the major muscle groups, including the quadriceps, gastrocnemius, biceps, triceps, trapezius, deltoid, and many others. Striated muscles tend to be very long, and many can function independently. However, some striated muscles are not attached to the skeleton, including those in the mouth, anus, heart, and upper esophagus.

[0086] Smooth muscle, on the other hand, has a very different structure. Rather than a series of long muscles with separate skeletal attachments, smooth muscle tends to be organized into continuous sheets with mechanical connections between smooth muscle cells. Smooth muscle is often located within the walls of hollow organs and is not usually under voluntary control. The smooth muscles lining a particular organ must support the same load and contract simultaneously. Smooth muscle functions, at least in part, to handle changes in load on hollow organs caused by movement and / or changes in position or pressure. This dual role means that smooth muscle must not only be able to contract like striated muscle, but also must be able to contract continuously to maintain organ dimensions against sustained loads. Examples of smooth muscle are those lining blood vessels, bronchioles, the bladder, and the gastrointestinal tract, such as the rectum.

[0087] Muscle strength depends on the number and size of muscle cells and their anatomical arrangement. An increase in muscle fiber diameter, either through the synthesis of new myofibrils (hypertrophy) and / or the formation of more muscle cells (thickening), increases the muscle's force-generating capacity.

[0088] Muscles can be classified by location or function. In some embodiments, the Flt-1 antibody or antigen-binding fragment thereof targets one or more facial muscles, one or more muscles of mastication, one or more tongue and neck muscles, one or more chest muscles, one or more shoulder girdle and arm muscles, one or more arm and shoulder muscles, one or more ventral and dorsal forearm muscles, one or more hand muscles, one or more erector spinae muscles, one or more pelvic girdle and leg muscles, and / or one or more front leg and foot muscles.

[0089] In some embodiments, facial muscles include, but are not limited to, intrinsic eye muscles such as the ciliary muscle, dilator pupillae, and sphincter iris; ear muscles such as the auricle, temporoparietal, stapedius, and tensor tympani; nose muscles such as the root of the nose, nasal muscles, dilator naris, depressor septum naris, and levator labii caudalis superioris; levator anguli oris, depressor anguli oris, orbicularis oris, buccinator, zygomaticus major and zygomaticus minor, platysma, levator labii superioris, depressor labii inferioris, laughing muscles, mentalis, and / or corrugator supercilii.

[0090] In some embodiments, muscles of mastication include, but are not limited to, masseter, temporalis, medial pterygoid, and lateral pterygoid. In some embodiments, muscles of the tongue and neck include, but are not limited to, genioglossus, styloglossus, palatoglossus, hyoglossus, digastric, stylohyoid, mylohyoid, geniohyoid, omohyoid, sternohyoid, sternothyroid, thyrohyoid, sternocleidomastoid, anterior scalene, middle scalene, and / or posterior scalene.

[0091] In some embodiments, muscles of the chest, pectoral girdle, and arms include, but are not limited to, the subclavius, pectoralis major, pectoralis minor, rectus abdominis, external oblique, internal oblique, transverse abdominis, diaphragm, external intercostal, internal intercostal, serratus anterior, trapezius, levator scapulae, rhomboid major, rhomboid minor, latissimus dorsi, deltoid, subscapularis, supraspinatus, infraspinatus, teres major, teres minor, and / or coracobrachialis.

[0092] In some embodiments, muscles of the arm and shoulder include, but are not limited to, the long head of the biceps, the short head of the biceps, the long head of the triceps, the lateral head of the triceps, the medial head of the triceps, the anconeus, the pronator teres, the supinator, and / or the brachialis.

[0093] In some embodiments, the ventral and dorsal forearm muscles include, but are not limited to, brachioradialis, flexor carpi radialis, flexor carpi ulnaris, palmaris longus, extensor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, and extensor digitorum minimi.

[0094] In some embodiments, the muscles of the hand include, but are not limited to, intrinsic muscles of the hand such as the thenar muscles, abductor pollicis brevis, flexor pollicis brevis, opponens pollicis, thenar muscles, abductor digiti minimi, flexor digiti minimi brevis, opponens digiti minimi, palmar interosseous, dorsal interosseous, and / or lumbrical muscles.

[0095] In some embodiments, erector spinae muscles include, but are not limited to, cervical muscles, spinalis, longissimus, and / or iliocostalis.

[0096] In some embodiments, muscles of the pelvic girdle and legs include, but are not limited to, psoas major, iliacus, quadratus femoris, adductor longus, adductor brevis, adductor magnus, gracilis, sartorius, rectus femoris, quadriceps femoris such as vastus lateralis, vastus medialis, and vastus intermedius, gastrocnemius, peroneus longus, soleus, gluteus maximus, gluteus medius, gluteus minimus, hamstrings: biceps femoris long head, hamstrings: biceps femoris short head, hamstrings: semitendinosus, hamstrings: semimembranosus, tensor fasciae latae, pubococcus, and / or tibialis anterior.

[0097] In some embodiments, muscles of the fore leg and foot include, but are not limited to, extensor digitorum longus, extensor pollicis longus, peroneus brevis, plantaris, tibialis posterior, flexor hallucis longus, extensor digitorum brevis, extensor pollicis brevis, abductor pollicis brevis, flexor digitorum brevis, abductor digitorum minimi, flexor digitorum brevis, opponens digitorum minimi, extensor digitorum brevis, lumbrical muscles of the foot, quadratus plantaris or flexor digitorum accessory, flexor digitorum brevis, dorsal interosseous, and / or plantar interosseous muscles.

[0098] Exemplary muscle targets are summarized in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0099] muscular dystrophy Muscular dystrophies are a group of genetic diseases that cause muscle degeneration, leading to weakness and impaired movement. The main characteristic of all muscular dystrophies is that they are progressive in nature. Muscular dystrophies include, but are not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, and myotonic dystrophy types 1 and 2, including congenital forms of myotonic dystrophy type 1. Symptoms may vary depending on the type of muscular dystrophy, with some or all muscles affected. Exemplary symptoms of muscular dystrophy include delayed development of muscle motor skills, difficulty using one or more muscle groups, difficulty swallowing, speaking, or eating and drinking, drooling, ptosis, frequent falls, decreased strength of a muscle or muscle group as an adult, decreased muscle size, difficulty walking due to body weakness or altered biomechanics, and / or cognitive or behavioral impairment / mental retardation.

[0100] Although there is no known cure for muscular dystrophies, several supportive therapies, including both symptomatic and disease-modifying treatments, are used. Corticosteroids, ACE inhibitors, angiotensin receptor blockers, physical therapy, orthotics, wheelchairs, or other assistive medical devices for ADL and pulmonary function are commonly used in muscular dystrophies. In myotonic dystrophy, cardiac pacemakers are used to prevent sudden death from cardiac arrhythmias. Antitonic drugs that improve the symptoms of myotonia (inability to relax) include mexiletine, and in some cases phenytoin, procainamide, and quinine.

[0101] Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is an X-linked recessive form of muscular dystrophy that results in muscle degeneration and eventual death. DMD is characterized by proximal muscle weakness, abnormal gait, hypertrophy of the gastrocnemius (lower leg) muscles, and elevated creatine kinase. Most DMD patients are diagnosed around age 5, at which point symptoms / signs usually become more apparent. Affected individuals typically lose the ability to walk around age 10-13 and die in or before their mid-to-late 20s due to respiratory complications and cardiomyopathy.

[0102] In individuals with DMD, serum creatine kinase concentrations can be increased by more than 10-fold compared to non-affected individuals. In some embodiments, administering a provided composition to an affected individual results in a decreased serum creatine kinase concentration compared to the baseline level before treatment. Typically, the baseline level is measured immediately before treatment. In some embodiments, administering a provided composition results in a serum creatine kinase concentration that is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to the baseline serum creatine kinase concentration immediately before treatment. In some embodiments, administering provided compositions results in a serum creatine kinase concentration that is reduced to less than about 3500 IU / L, 3000 IU / L, 2500 IU / L, 2000 IU / L, 1500 IU / L, 1000 IU / L, 750 IU / L, 500 IU / L, 250 IU / L, 100 IU / L, 90 IU / L, 80 IU / L, 70 IU / L, or 60 IU / L. In some embodiments, administering provided compositions results in a reduced serum creatine kinase concentration compared to the serum creatine kinase concentration of an untreated subject.

[0103] The disease DMD is caused by mutations in the dystrophin gene, located on the human X chromosome, which encodes the protein dystrophin, a key structural component in muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. Dystrophin connects the internal cytoplasmic actin filament network and the extracellular matrix, providing physical strength to muscle fibers. Thus, alteration or absence of dystrophin leads to abnormal sarcolemmal tearing and muscle fiber necrosis. While both men and women can carry mutations, women rarely show severe symptoms of the disease.

[0104] The primary symptom of DMD is muscle weakness associated with muscle wasting, usually with voluntary muscles being affected first, particularly affecting the hips, pelvic region, thighs, shoulder muscles, and lower leg muscles. Muscle weakness also occurs in the arms, neck, and other areas. The lower legs are often swollen. Signs and symptoms usually appear before age 6 and may appear as early as infancy. Cardiomyopathy usually occurs in individuals with DMD after age 18. Other physical symptoms include, but are not limited to, delayed ability to ambulate independently, progressive difficulty walking, stepping, or running, and eventual loss of the ability to ambulate (usually by age 12), frequent falls, fatigue, difficulty with motor functions (running, hopping, jumping), increased lumbar lordosis leading to shortening of the hip flexors, dysfunction of the Achilles tendon and hamstring muscles, pseudohypertrophy (swelling) of the tongue and calf muscles caused by connective tissue fibrosis, muscle fiber deformation, replacement of muscle fibers with fat and connective tissue, neurobehavioral disorders (e.g., ADHD), learning disabilities (dyslexia), and a higher risk of non-progressive decline in certain cognitive functions (particularly short-term verbal memory), and skeletal deformities (including scoliosis in some cases).

[0105] Muscle changes seen in DMD involve an increase in connective tissue, i.e., the development of fibrosis, as well as resulting from reactive or repair processes involving mechanical, humoral, and / or cellular factors. Lack of functional dystrophin leads to instability of the sarcolemma, making cells less tolerant to mechanical shear forces and prone to electrolyte overload, leading to tissue breakdown. When DMD muscle tissue is damaged, recovery is limited by the ability of satellite cells to proliferate, resulting in necrosis, inflammation, fibrosis, and adipocyte replacement. Increased connective tissue occurs early in the disease process, due to an increase in loose connective tissue covering the sarcolemma (i.e., endomysium), which sheathes each muscle cell, before observable muscle damage. Increased collagenous connective tissue negatively impacts nutrient supply to affected muscle cells and contributes to DMD muscle pathology, secondary to the effects of locomotor strength and its loss with age.

[0106] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof reduces fibrosis in muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is cardiac muscle, diaphragm muscle, gastrocnemius muscle, and / or tibialis anterior (TA) muscle. In some embodiments, reduced fibrosis is indicated by reduced collagen staining. In some embodiments, the collagen is type I collagen. In some embodiments, reduced fibrosis can be determined, for example, by measuring the % collagen-positive area in the muscle of a mouse administered with an anti-Flt-1 antibody or antigen-binding fragment thereof. For example, the % collagen-positive area in the diaphragm muscle of a mouse administered with an anti-Flt-1 antibody or antigen-binding fragment thereof can be at least about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, or about 9.0% of the total tissue area. In certain embodiments, the % collagen-positive area in the diaphragm muscle of mice administered an anti-Flt-1 antibody can be significantly lower than the % collagen-positive area in the diaphragm muscle of mice administered an isotype control antibody.

[0107] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof reduces necrosis of muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is cardiac muscle, diaphragm muscle, gastrocnemius muscle, and / or tibialis anterior (TA) muscle. In some embodiments, reduced necrosis can be determined, for example, by measuring the % necrosis-positive area in the muscle of a mouse administered with an anti-Flt-1 antibody or antigen-binding fragment thereof. For example, the % necrosis-positive area in the diaphragm muscle of a mouse administered with an anti-Flt-1 antibody or antigen-binding fragment thereof can be at least about 0.5%, about 0.45%, about 0.4%, about 0.35%, about 0.3%, about 0.25%, about 0.2%, about 0.15%, about 0.1%, about 0.05%, or about 0.025% of the total tissue area. In certain embodiments, the % necrotic area in the gastrocnemius muscle of mice administered an anti-Flt-1 antibody can be significantly lower than the % necrotic area in the gastrocnemius muscle of mice administered an isotype control antibody.

[0108] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in increased muscle strength and / or resistance to fatigue.

[0109] Flt-1 receptor The Flt-1 receptor, also known as vascular endothelial growth factor receptor 1 (VEGFR-1) or Flt-1, is encoded by the FLT1 gene and expressed on the plasma membrane of endothelial cells and monocytes. The vascular endothelial growth factor (VEGF) family of signaling glycoproteins acts as a potent promoter of angiogenesis during embryonic and postnatal development. Specifically, binding of the VEGF-A ligand to the VEGF receptor has been shown to promote vascular permeability and further induce the migration, proliferation, and survival of endothelial cells, which then provide the basic structure for the new vasculature. The dominant VEGF signaling molecule for angiogenesis (VEGF-A) mediates its signaling through VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). Soluble forms of Flt-1 (sFlt-1) also exist, but lack the intracellular signaling domain and are therefore thought to play only a role in regulatory capacities by sequestering VEGF-A or other ligands that bind to it. sFlt-1 and other molecules containing Flt-1 binding sites that are not linked to intracellular signaling pathways are called "decoy receptors." Flt-1 and Flk-1 receptors contain an extracellular VEGF-A binding domain and an intracellular tyrosine kinase domain, and both are expressed during development of angioblast and endothelial cell lineages and during tissue regeneration. Flt-1, compared to Flk-1, exhibits a greater ability to bind VEGF-A (K d about 2 -10VEGF-A has approximately 10-fold greater binding affinity (pM) but weaker tyrosine kinase activity, indicating that the angiogenic signaling following VEGF-A binding to Flt-1 is relatively weaker than that resulting from VEGF-A binding to Flk-1. Therefore, homozygous Flt-1 gene knockout mice die fetally from endothelial cell overproduction and vascular disorganization. Conversely, homozygous Flk-1 gene knockout mice die due to impaired development of organized blood vessels due to a lack of yolk sac blood island formation during embryogenesis. Both Flt-1 and Flk-1 receptors are required for normal development. However, selectively increasing VEGF-A concentration can allow greater binding to Flk-1 receptors, eliciting proangiogenic effects that increase capillary density and facilitate muscle regeneration, reduce fibrosis and inflammation, and alleviate symptoms and features associated with DMD and other muscular dystrophies in various muscle tissues.

[0110] As used herein, the term "Flt-1 receptor" refers to both soluble and membrane-bound Flt-1 receptor or functional fragments thereof.

[0111] Anti-Flt-1 antibody As used herein, the term "anti-Flt-1 antibody" refers to any antibody or antigen-binding fragment thereof that binds to the Flt-1 receptor (e.g., soluble or membrane-bound Flt-1 receptor). In some embodiments, an anti-Flt-1 antibody is generated that binds to the Flt-1 receptor with high affinity. Without being bound by theory, it is believed that binding of the anti-Flt-1 antibody to the Flt-1 receptor inhibits one or more endogenous ligands from binding to Flt-1, thereby allowing a greater amount of available ligand to associate with other VEGF receptors, such as the Flk-1 receptor. Increased VEGF availability promotes angiogenesis with increased blood flow to muscles, combating functional ischemia and improving the structural and functional characteristics of DMD. In some embodiments, binding of the antibody to the Flt-1 receptor increases the amount of VEGF available to bind to other VEGF receptors.

[0112] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a sequence provided in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14]

[0113] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL CDR1 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 19 to 21; a VL CDR2 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 22 to 24; a VL CDR3 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 25 to 34; The VL CDR3 comprises one or more complementarity determining regions (CDRs) selected from the group consisting of a VH chain CDR1 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 1-4, a VH CDR2 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 5-14, and a VH CDR3 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 15-18. In some embodiments, the VL CDR3 is not SEQ ID NO: 25. In some embodiments, the VH CDR3 is not SEQ ID NO: 15.

[0114] In some embodiments, one or more CDRs comprise a VL CDR3 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 25-34, and a VH CDR3 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 15-18. In some embodiments, the VL CDR3 is not SEQ ID NO: 25. In some embodiments, the VH CDR3 is not SEQ ID NO: 15.

[0115] In some embodiments, the one or more CDRs comprise a VL CDR1 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 19-21, a VL CDR2 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 22-24, and a VL CDR3 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity to any one of SEQ ID NOs: 25-34. In some embodiments, the VL CDR3 is not SEQ ID NO: 25.

[0116] In some embodiments, the one or more CDRs comprise a VH CDR1 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 1-4, a VH CDR2 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 5-14, and a VH CDR3 defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95% or 98% identity to any one of SEQ ID NOs: 15-18. In some embodiments, the VH CDR3 is not SEQ ID NO: 15.

[0117] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain comprising a VL CDR1, a VL CDR2, and a VL CDR3 defined by the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain comprising a VL CDR1, a VL CDR2, and a VL CDR3 defined by the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 23, and SEQ ID NO: 25, respectively. In yet another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain comprising a VL CDR1 and a VL CDR2 defined by the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 24, respectively, and a VL CDR3 defined by the amino acid sequence of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34. In certain embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VL chain comprising a VL CDR1 defined by the amino acid sequence of SEQ ID NO: 21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO: 24, and a VL CDR3 defined by the amino acid sequence of SEQ ID NO: 32. In some embodiments, the VL CDR3 is not SEQ ID NO: 25.

[0118] In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 15, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain comprising a VH CDR1, a VH CDR2, and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:16, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain comprising a VH CDR1, a VH CDR2, and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:18, respectively. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain comprising a VH CDR1 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:17, respectively, and a VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH CDR1 and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:17, respectively, and a VH CDR1 and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:12. In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain comprising a VH CDR1, a VH CDR2, and a VH CDR3 defined by the amino acid sequences of SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:17, respectively. In a specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a VH chain comprising a VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17. In some embodiments, the VH CDR3 is not SEQ ID NO:15.

[0119] In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a light chain VL region comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, or 98% identical to any one of SEQ ID NOs: 49 to 61, and / or a heavy chain VH region comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, or 98% identical to any one of SEQ ID NOs: 35 to 48. In a specific embodiment, the VL region comprises the amino acid sequence of SEQ ID NO: 60, and the VH region comprises the amino acid sequence of SEQ ID NO: 45. In another embodiment, the antibody further comprises a heavy chain constant region comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, or 98% identical to any one of SEQ ID NOs: 87 to 89. In some embodiments, the VL region is not SEQ ID NO: 49 or SEQ ID NO: 50. In some embodiments, the VH region is not SEQ ID NO: 35 or SEQ ID NO: 36.

[0120] In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a light chain comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, or 98% identical to any one of SEQ ID NOs: 75 to 86, and / or a heavy chain comprising an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, or 98% identical to any one of SEQ ID NOs: 62 to 74. In a specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO: 76, and the heavy chain region comprises the amino acid sequence of SEQ ID NO: 71.

[0121] In some embodiments, the heavy chain of the anti-Flt-1 antibody or antigen-binding fragment thereof has the amino acid sequence MGWSCIILFLVATATGVHSELQLVESGGGLVQPGGSLRLSCAASGFTFSDYSASWVRQAPGKGLEWVSAISWSGDSTYYAESVKGRFTIFRDNSKNTLYLQMNSLRAEDTAVYYCAKSWATP IESLYYYGSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP In some embodiments, the heavy chain of an anti-Flt-1 antibody or antigen-binding fragment thereof comprises the amino acid sequence: KSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKX (SEQ ID NO: 108). ELQLVESGGGLVQPGGSLRLSCAASGFTFSDYSASWVRQAPGKGLEWVSAISWSGDSTYYAESVKGRFTIFRDNSKNTLYLQMNSLRAEDTAVYYCAKSWATPIESLYYYGSDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109).

[0122] In some embodiments, the light chain of the anti-Flt-1 antibody or antigen-binding fragment thereof has the amino acid sequence MGWSCIILFLVATATGVHSSYELTQPLSVSVALRQAAKITCGGNNIGSQTAQWYQQKPGQAPVLVIYANNRRPSGIPERFSGSKSGNTATLTISRAQAGDEADYYCQVWDASTQAIVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSX (SEQ ID NO: 110).

[0123] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO:108 or SEQ ID NO:109 and a light chain of SEQ ID NO:110 or SEQ ID NO:76.

[0124] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is -7 Super M, about 0.5×10 -7 Over M, about 10 -8 Super M, about 0.5×10 -8 Over M, about 10 -9 Super M, about 0.5×10 -9 Over M, about 10 -10 Super M, about 0.5×10 -10 Over M, about 10 -11 Super M, about 0.5×10 -11 Over M, about 10 -12 More than M or about 0.5 × 10 -12 In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 with an affinity of greater than about 10 M. -7 Super M, about 0.5×10 -7 Over M, about 10 -8 Super M, about 0.5×10 -8 Over M, about 10 -9 Super M, about 0.5×10 -9 Over M, about 10 -10 Super M, about 0.5×10 -10 Over M, about 10 -11 Super M, about 0.5×10 -11 Over M, about 10 -12 More than M or about 0.5 × 10 -12 It binds to mouse Flt-1 with an affinity greater than M. The affinity of an Flt-1 antibody can be measured by a surface plasmon resonance assay, for example, a BIACORE assay.

[0125] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has an IC of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM in a competition assay with human Flt-1. 50In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has an IC of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM in a competition assay with mouse Flt-1. 50 It is characterized by:

[0126] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof inhibits the binding and / or activity of VEGF at the Flt-1 receptor. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has an IC of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 50 pM, less than about 25 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM for inhibiting VEGF binding to human Flt-1 in a competition assay. 50 It is characterized by:

[0127] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof competes with and / or inhibits the binding of VEGF to soluble Flt-1. In other embodiments, the competition and / or inhibition is dose-dependent. In certain embodiments, the inhibition of VEGF binding to Flt-1 results in increased phosphorylation of VEGF R2. Without being bound by theory, the binding of the anti-Flt-1 antibody or antigen-binding fragment thereof to Flt-1 inhibits VEGF binding to Flt-1. Unbound VEGF binds to VEGF R2, which can be demonstrated by measuring the phosphorylation of VEGF R2. In certain embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof rescues the phosphorylation of VEGF R2 in a dose-dependent manner. For example, phosphorylation of VEGF R2 may be rescued by at least about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10% or about 5%.

[0128] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof provides greater than about 95%, greater than about 90%, greater than about 85%, greater than about 80%, greater than about 75%, greater than about 70%, greater than about 65%, greater than about 60%, greater than about 55%, greater than about 50%, greater than about 45%, greater than about 40%, greater than about 35%, greater than about 30%, greater than about 25%, greater than about 20%, greater than about 15%, or greater than about 10% rescue in a bioassay. In certain embodiments, the bioassay involves human vein endothelial cells (HUVECs) stimulated with VEGF in the presence of sFlt-1 and the anti-Flt-1 antibody or antigen-binding fragment thereof. VEGF-induced activation of the cells can be tested by determining the phosphorylation status of the VEGF R2 receptor. Data can be expressed as % rescue of VEGF R2 receptor phosphorylation relative to VEGF R2 receptor phosphorylation in the presence of sFlt-1 alone (eg, without anti-Flt-1 antibody).

[0129] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a half-life of greater than about 200 hours, greater than about 150 hours, greater than about 100 hours, greater than about 95 hours, greater than about 90 hours, greater than about 85 hours, greater than about 80 hours, greater than about 75 hours, greater than about 70 hours, greater than about 65 hours, greater than about 60 hours, greater than about 55 hours, greater than about 50 hours, or greater than about 45 hours, and ranges therein. In some embodiments, the half-life is measured in mice.

[0130] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a maximum serum concentration of greater than about 400 μg / mL, greater than about 375 μg / mL, greater than about 350 μg / mL, greater than about 325 μg / mL, greater than about 300 μg / mL, greater than about 275 μg / mL, greater than about 250 μg / mL, greater than about 225 μg / mL, greater than about 200 μg / mL, greater than about 175 μg / mL, greater than about 150 μg / mL, greater than about 125 μg / mL, greater than about 100 μg / mL, greater than about 75 μg / mL, or greater than about 50 μg / mL, and ranges therein. In some embodiments, the maximum serum concentration is measured in mice.

[0131] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to Flt-1 with minimal or weak binding to other VEGF receptors, hi some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to Flt-1 with minimal or weak binding to VEGF R2 (Flk-1) and / or VEGF R3 (Flt-4).

[0132] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has an affinity for human Flt-1 of about 1×10 -3 M -1 seconds -1 Super, about 1×10 -4 M -1 seconds -1 Super, about 1×10 -5 M -1 seconds -1 Super, about 1×10 -6 M -1 seconds -1 greater than, or approximately 1 × 10 -7 M -1 seconds -1 It has super ka.

[0133] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has an affinity for human Flt-1 of about 1×10 -3 seconds -1 Super, about 1×10 -4 seconds -1 Super, about 1×10 -5 seconds -1 greater than, or approximately 1 × 10 -6 seconds -1 It has a kd of over 100.

[0134] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has an affinity for human Flt-1 of about 1×10 -8 Super M, about 1×10 -9 Super M, about 1×10 -10 Super M, about 1×10 -11 More than M or about 1 × 10 -12 K over M D It has.

[0135] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to soluble Flt-1. In certain embodiments, the binding is dose-dependent, such that higher concentrations of the antibody or antigen-binding fragment thereof bind to greater amounts of soluble Flt-1.

[0136] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a percent human identity of greater than about 99%, greater than about 98%, greater than about 97%, greater than about 96%, greater than about 95%, greater than about 94%, greater than about 93%, greater than about 92%, greater than about 91%, greater than about 90%, or greater than about 80%.

[0137] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a percent human homology of greater than about 99%, greater than about 98%, greater than about 97%, greater than about 96%, greater than about 95%, greater than about 94%, greater than about 93%, greater than about 92%, greater than about 91%, greater than about 90%, or greater than about 80%.

[0138] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to Flt-1 protein. In some embodiments, the Flt-1 protein is a recombinant protein, e.g., recombinant sFlt-1. In a specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 1 (NP_002010.2 GI:156104876, SEQ ID NO:90) (Table 13). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform X1 (XP_01153316.1 GI:767977511, SEQ ID NO:91). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 2 precursor (NP_001153392.1 GI:229892220, SEQ ID NO:92). In yet another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 3 precursor (NP_001153502.1 GI:229892300, SEQ ID NO: 93). In another embodiment, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 isoform 4 precursor (NP_001153503.1 GI:229892302, SEQ ID NO: 94).

[0139] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to a specific epitope of the Flt-1 protein, for example, the anti-Flt-1 antibody or antigen-binding portion thereof binds to an amino acid sequence as set forth in Table 3. [Table 3]

[0140] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in a peak serum antibody concentration of at least about 700 μg / mL, at least about 650 μg / mL, at least about 600 μg / mL, at least about 550 μg / mL, at least about 500 μg / mL, at least about 450 μg / mL, at least about 400 μg / mL, at least about 350 μg / mL, at least about 300 μg / mL, at least about 250 μg / mL, at least about 200 μg / mL, at least about 150 μg / mL, at least about 100 μg / mL, at least about 50 μg / mL, at least about 40 μg / mL, at least about 30 μg / mL, at least about 20 μg / mL, at least about 10 μg / mL, or at least about 5 μg / mL, and ranges therein. In some embodiments, the peak serum antibody concentration is dose-dependent.

[0141] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in a minimum serum antibody concentration of at least about 450 μg / mL, at least about 400 μg / mL, at least about 350 μg / mL, at least about 300 μg / mL, at least about 250 μg / mL, at least about 200 μg / mL, at least about 150 μg / mL, at least about 100 μg, at least about 50 μg / mL, or at least about 25 μg / mL, and ranges therein. In some embodiments, the minimum serum antibody concentration is dose-dependent.

[0142] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in a decreased serum concentration of soluble Flt-1 compared to baseline levels or compared to levels in subjects administered vehicle alone. Baseline levels are typically measured immediately prior to administration. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in a decreased serum concentration of soluble Flt-1 by at least about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% compared to the baseline serum concentration of soluble Flt-1 immediately prior to administration. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof provides a serum FLT-1 antibody or antigen-binding fragment thereof of less than about 4000 pg / mL, about 3500 pg / mL, about 3000 pg / mL, about 2500 pg / mL, about 2000 pg / mL, about 1750 pg / mL, about 1500 pg / mL, about 1250 pg / mL, about 1000 pg / mL, about 900 pg / mL, about 800 pg / mL, about The administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in a decreased serum concentration of soluble Flt-1 of about 700 pg / mL, about 600 pg / mL, about 500 pg / mL, about 450 pg / mL, about 400 pg / mL, about 350 pg / mL, about 300 pg / mL, about 250 pg / mL, about 200 pg / mL, about 150 pg / mL, about 100 pg / mL, about 50 pg / mL, or about 10 pg / mL, and ranges thereof. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in a decreased serum concentration of soluble Flt-1 compared to the serum concentration of soluble Flt-1 in a subject not administered the antibody or antigen-binding fragment thereof. In some embodiments, the decreased serum concentration of soluble Flt-1 is dose-dependent.

[0143] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increased serum concentration of VEGF compared to baseline levels or compared to levels in subjects administered vehicle alone. Typically, baseline levels are measured immediately prior to treatment. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increased serum concentration of VEGF of at least about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% compared to the baseline serum concentration of VEGF immediately prior to administration. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increased serum concentration of VEGF of greater than about 500 pg / mL, about 450 pg / mL, about 400 pg / mL, about 350 pg / mL, about 300 pg / mL, about 250 pg / mL, about 200 pg / mL, about 150 pg / mL, about 100 pg / mL, about 50 pg / mL, or about 25 pg / mL, and ranges therein. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in an increased serum concentration of VEGF compared to the serum concentration of VEGF in an untreated subject. In some embodiments, the increased serum concentration of VEGF is dose-dependent.

[0144] In some embodiments, in vivo administration of an anti-Flt-1 antibody or antigen-binding fragment thereof results in increased vascularization of muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is diaphragm, gastrocnemius, and / or tibialis anterior (TA) muscle. In some embodiments, increased angiogenesis is indicated by increased CD31 staining of an endothelial cell marker, e.g., CD31. In some embodiments, increased staining can be determined, for example, by measuring the % CD31-positive area in muscle of mice administered the anti-Flt-1 antibody or antigen-binding fragment thereof. For example, the CD31-positive area% in the diaphragm muscle of a mouse administered an anti-Flt-1 antibody or antigen-binding fragment thereof can be at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% of the total tissue area. In a further example, the CD31-positive area% in the TA muscle of a mouse administered an anti-Flt-1 antibody or antigen-binding fragment thereof can be at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% of the total tissue area. In certain embodiments, the % CD31-positive area in the diaphragm or TA muscle of mice administered an anti-Flt-1 antibody may be significantly higher than the % CD31-positive area in the diaphragm or TA muscle of mice administered an isotype control antibody.

[0145] In some embodiments, increased staining of endothelial cell markers can be measured, for example, by measuring the normalized % CD31 positivity in muscles of mice administered an anti-Flt-1 antibody or antigen-binding fragment thereof. In certain embodiments, the increased CD31 staining in muscles of mice administered an anti-Flt-1 antibody or antigen-binding fragment thereof is compared to the CD31 staining measured in muscles of mice administered an isotype control antibody. For example, the normalized % CD31 positivity in diaphragm muscles of mice administered an anti-Flt-1 antibody or antigen-binding fragment thereof can be at least about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, or about 110%. In further examples, the normalized CD31 positivity % of the TA muscle of mice administered an anti-Flt-1 antibody or antigen-binding fragment thereof can be at least about 300%, about 290%, about 280%, about 270%, about 260%, about 250%, about 240%, about 230%, about 220%, about 210%, about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, or about 110%, and ranges therein.

[0146] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to human Flt-1 and has minimal or weak binding to Flt-1 receptors of other mammals (e.g., 10 -7 M or 10 -6 with a binding affinity of less than M. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to human Flt-1 and does not bind to monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof selectively binds to human Flt-1 and does not bind to mouse Flt-1.

[0147] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 and monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to cynomolgus monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to human Flt-1 and mouse Flt-1.

[0148] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is selected from the group consisting of an IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, a diabody, a triabody, and a tetrabody.

[0149] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is an IgG. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is an IgG1.

[0150] Engineered constant regions In some embodiments, a suitable anti-Flt-1 antibody contains an Fc domain or a portion thereof that binds to the FcRn receptor. As a non-limiting example, a suitable Fc domain may be derived from an immunoglobulin subclass, such as IgG. In some embodiments, a suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. Particularly suitable Fc domains include those derived from human or humanized antibodies.

[0151] It is contemplated that improved binding between the Fc domain and the FcRn receptor results in a prolonged serum half-life. Thus, in some embodiments, a suitable Fc domain (SEQ ID NO: 104) comprises one or more amino acid mutations that lead to improved binding to FcRn. Various mutations in Fc domains that result in improved binding to FcRn are well known in the art and can be adapted to the practice of the present invention. In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to Leu234, Ler235, Gly237, Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and / or Asn434 of human IgG1.

[0152] Some mutations in the Fc domain result in reduced binding of IgG to the FcRn receptor, thereby inhibiting effector function. In some embodiments, a suitable Fc domain (SEQ ID NO: 104) contains one or more mutations at one or more positions corresponding to Leu234, Ler235, and Gly237 of human IgG1. In a specific embodiment, Leu234 is mutated to Ala. In another embodiment, Leu235 is mutated to Ala. In yet another embodiment, Gly237 is mutated to Ala.

[0153] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof contains a spacer and / or is linked to another entity. In some embodiments, the linker or spacer is [ka] (SEQ ID NO: 105) (GAG linker). In some embodiments, the linker or spacer comprises a sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to [ka] (SEQ ID NO: 106) (GAG2 linker). In some embodiments, the linker or spacer comprises a sequence at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to (SEQ ID NO: 106) (GAG2 linker). [ka] (SEQ ID NO: 107) (GAG3 linker).

[0154] Generation of anti-Flt-1 antibodies and antigen-binding fragments Suitable recombinant anti-Flt-1 antibodies or antigen-binding fragments thereof of the present invention can be produced by any available method, for example, recombinant anti-Flt-1 antibodies or antigen-binding fragments can be produced recombinantly by utilizing host cell lines engineered to express nucleic acids encoding the recombinant anti-Flt-1 antibodies or antigen-binding fragments.

[0155] Thus, the present invention further provides polynucleotide sequences encoding the various amino acid sequences described herein. In some embodiments, the present invention provides polynucleotide sequences encoding the anti-Flt-1 antibody heavy or light chain amino acid sequences described herein, e.g., any one of SEQ ID NOS: 62-86 or 108-110. In some embodiments, the present invention provides polynucleotide sequences encoding the variable regions of the anti-Flt-1 antibody heavy or light chain amino acid sequences described herein, e.g., any one of SEQ ID NOS: 35-61. In some embodiments, the present invention provides polynucleotide sequences encoding the CDR regions of the anti-Flt-1 antibody heavy or light chain amino acid sequences described herein, e.g., any one of SEQ ID NOS: 1-34. In some embodiments, the present invention provides polynucleotide sequences encoding the anti-Flt-1 antibody constant region amino acid sequences described herein, e.g., any one of SEQ ID NOS: 87-89. In some embodiments, the present invention provides polynucleotide sequences encoding the anti-Flt-1 antibody Fc region amino acid sequences described herein, e.g., SEQ ID NOS: 104. In some embodiments, the present invention provides polynucleotide sequences encoding the anti-Flt-1 antibody linker amino acid sequences described herein, e.g., SEQ ID NOS: 105-107.

[0156] In some embodiments, the polynucleotide sequence encoding the anti-Flt-1 antibody heavy chain, light chain, variable region, CDR region, Fc region, or linker region amino acid sequence further comprises a sequence encoding a signal peptide. As a non-limiting example, a suitable signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 111).

[0157] The various polynucleotide sequences described herein can be embodied in a variety of vector systems for expression of recombinant anti-Flt-1 antibodies or antigen-binding fragments thereof.

[0158] If the antibody is produced recombinantly, any expression system can be used, including, for example, egg, baculovirus, plant, yeast, or mammalian cells, to name but a few known expression systems.

[0159] In some embodiments, recombinant anti-Flt-1 antibodies or antigen-binding fragments suitable for the present invention are produced in mammalian cells. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include the BALB / c mouse myeloma line (NSO / 1, ECACC No.: 85110503), human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands), and SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651).

[0160] In some embodiments, the present invention provides a recombinant anti-Flt-1 antibody or antigen-binding fragment thereof produced from a human cell. In some embodiments, the present invention provides a recombinant anti-Flt-1 antibody or antigen-binding fragment thereof produced from a CHO cell.

[0161] Pharmaceutical compositions containing the antibodies of the present invention The present invention further provides pharmaceutical compositions comprising a therapeutically active ingredient according to the invention (e.g., an anti-Flt-1 antibody or antigen-binding fragment thereof) together with one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions can optionally include one or more additional therapeutically active substances.

[0162] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to make the compositions suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or perform such modifications, if necessary, with no more than routine experimentation.

[0163] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association the active ingredient with a diluent or other excipient or carrier, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0164] Pharmaceutical compositions according to the present invention can be prepared, packaged, and / or sold in bulk as single unit doses and / or multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dose, for example, 1 / 2 or 1 / 3 of such a dose.

[0165] The relative amounts of active ingredient, pharmaceutically acceptable excipient or carrier, and / or any additional ingredients in a pharmaceutical composition according to the invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain between 0.1% and 100% (w / w) active ingredient.

[0166] Pharmaceutical compositions may additionally contain pharmaceutically acceptable excipients or carriers, which as used herein includes any solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21 stEdition, AR Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006, incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and well-known techniques for preparing them. Except insofar as any conventional excipient vehicle or carrier is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effect or by otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0167] In some embodiments, a pharmaceutically acceptable excipient or carrier is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient or carrier is approved for human use and for veterinary use. In some embodiments, the excipient or carrier is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient and carrier are pharmaceutical grade. In some embodiments, the excipient or carrier meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0168] Pharmaceutically acceptable excipients or carriers used in the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Such excipients or carriers can be included in the pharmaceutical composition as desired. Excipients or carriers (e.g., cocoa butter and suppository waxes, coloring agents, coating agents, sweetening agents, flavoring agents, and / or perfuming agents) can be present in the composition according to the judgment of the formulator.

[0169] Suitable pharmaceutically acceptable excipients or carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose, or starch), sugars (e.g., mannitol, sucrose, etc.), dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc., and combinations thereof. If desired, the pharmaceutical preparation can be mixed with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavorings, and / or aromatic substances) that do not adversely react with or interfere with the activity of the active compound. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0170] Suitable pharmaceutical compositions or preparations can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents, if desired.The compositions can be solutions, suspensions, emulsions, tablets, pills, capsules, sustained-release formulations, or powders.The compositions can also be prepared as suppositories with conventional binders or carriers, such as triglycerides.Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0171] Pharmaceutical compositions or medicaments can be formulated in a conventional manner as pharmaceutical compositions suitable for administration to humans. For example, in some embodiments, compositions for intravenous administration are typically aqueous solutions in sterile isotonic buffer. If necessary, the composition may further include a solubilizing agent and a local anesthetic to alleviate pain at the injection site. Typically, the ingredients are supplied separately or mixed together in unit-dose form, e.g., as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water, saline, or dextrose / water. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0172] General information on the compounding and / or manufacturing of pharmaceuticals is available, for example, from Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (hereby incorporated by reference).

[0173] Route of administration The anti-Flt-1 antibodies or antigen-binding fragments thereof described herein (or compositions or medicaments containing the anti-Flt-1 antibodies or antigen-binding fragments thereof described herein) are administered by any suitable route. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment protein, or pharmaceutical compositions containing the same, are administered parenterally. Parenteral administration can be intravenous, intradermal, intrathecal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, and / or transmucosal. In some embodiments, the anti-Flt-1 antibodies or antigen-binding fragments thereof, or pharmaceutical compositions containing the same, are administered subcutaneously. As used herein, the term "subcutaneous tissue" is defined as the layer of loose, irregular connective tissue immediately beneath the skin. For example, subcutaneous administration can be performed by injecting the composition into areas including, but not limited to, the thigh, abdomen, buttocks, or scapular region. In some embodiments, the anti-Flt-1 antibodies or antigen-binding fragments thereof, or pharmaceutical compositions containing the same, are administered intravenously. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof, or pharmaceutical composition containing same, is administered orally. If desired, two or more routes can be used simultaneously.

[0174] In some embodiments, administration results in only a localized effect in an individual, while in other embodiments, administration results in an effect across multiple parts of an individual, e.g., a systemic effect. Typically, administration results in delivery of the anti-Flt-1 antibody or antigen-binding fragment to one or more target tissues, including, but not limited to, cardiac muscle, kidney, including striated and smooth muscle, liver, brain, spinal cord, intestinal tract, eye, lung, spleen, and heart.

[0175] In some embodiments, the striated muscle is selected from the group consisting of triceps, tibialis anterior, soleus, gastrocnemius, quadriceps, and diaphragm.

[0176] In some embodiments, smooth muscle is muscle lining blood vessels, bronchioles, the bladder, and the gastrointestinal tract, such as the rectum.

[0177] Dosage Forms and Dosage Regimen In some embodiments, the compositions are administered in a therapeutically effective amount and / or according to a dosing regimen that is associated with a particular desired outcome (e.g., treating or reducing the risk of a muscular dystrophy, such as Duchenne muscular dystrophy).

[0178] The specific dosage or amount administered in accordance with the present invention may vary depending, for example, on the nature and / or extent of the desired outcome, the details of the route and / or timing of administration, and / or one or more characteristics (e.g., body weight, age, medical history, genetic characteristics, lifestyle parameters, severity of cardiac defect and / or level of risk for cardiac defect, etc., or a combination thereof). Such dosage or amount can be determined by one of ordinary skill in the art. In some embodiments, an appropriate dosage or amount is determined according to standard clinical techniques. Alternatively, or additionally, in some embodiments, an appropriate dosage or amount is determined by the use of one or more in vitro or in vivo assays to help identify a desirable or optimal dosage range or amount to be administered.

[0179] In various embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is administered in a therapeutically effective amount. Generally, a therapeutically effective amount is sufficient to achieve a meaningful effect in a subject (e.g., treating, modulating, curing, preventing, and / or alleviating the underlying disease or condition). In some specific embodiments, appropriate dosages or amounts to be administered can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0180] In some embodiments, provided compositions are provided as pharmaceutical formulations, hi some embodiments, the pharmaceutical formulations are or comprise a unit dose amount for administration according to a dosing regimen that correlates with achieving a reduced incidence or risk of muscular dystrophy, such as Duchenne muscular dystrophy.

[0181] In some embodiments, a formulation comprising an anti-Flt-1 antibody or antigen-binding fragment described herein is administered as a single dose. In some embodiments, a formulation comprising an anti-Flt-1 antibody or antigen-binding fragment described herein is administered at regular intervals. As used herein, administration at "intervals" refers to the administration of a therapeutically effective amount on a regular basis (as distinguished from a single dose). The intervals can be determined by standard clinical techniques. In some embodiments, a formulation comprising an anti-Flt-1 antibody or antigen-binding fragment described herein is administered bimonthly, monthly, twice monthly, every three weeks, every other week, weekly, twice weekly, three times weekly, daily, twice daily, or every six hours. The administration interval for an individual need not be a fixed interval but can vary over time depending on the individual's needs. In certain embodiments, an anti-Flt-1 antibody or antigen-binding fragment thereof is administered twice weekly.

[0182] As used herein, the term "bimonthly" means administration once every two months (i.e., once every two months), the term "monthly" means administration once per month, the term "every three weeks" means administration once every three weeks (i.e., once every three weeks), the term "biweekly" means administration once every two weeks (i.e., once every two weeks), the term "weekly" means administration once per week, and the term "daily" means administration once per day.

[0183] In some embodiments, a formulation comprising an anti-Flt-1 antibody or antigen-binding fragment described herein is administered at regular intervals indefinitely. In some embodiments, a formulation comprising an anti-Flt-1 antibody or antigen-binding fragment described herein is administered at regular intervals for a predetermined period of time.

[0184] As used herein, the term "therapeutically effective amount" is primarily determined based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. The therapeutically effective amount is generally administered in a dosage regimen comprising multiple unit doses. For a particular composition, the therapeutically effective amount (and / or the appropriate unit dose within the effective dosage regimen) may vary depending on, for example, the route of administration and the combination with other pharmaceutical agents.

[0185] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is administered at a dosage ranging from about 0.1 mg / kg to about 50 mg / kg. In other embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is administered at a dose ranging from about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 3 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1.0 mg / kg to about 40 mg / kg, about 1.0 mg / kg to about 30 mg / kg, about 1.0 mg / kg to about 20 mg / kg, about 1.0 mg / kg to about 10 mg / kg, about 1.0 mg / kg to about 5 mg / kg, or about 1.0 mg / kg to about 3 mg / kg. In certain embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is administered at a dosage of about 1.0 mg / kg, 3.0 mg / kg, 10 mg / kg, or about 20 mg / kg.

[0186] In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof reduces the intensity, severity, or frequency of, or delays the onset of, at least one sign or symptom of DMD. In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof reduces the intensity, severity, or frequency of, or delays the onset of, at least one sign or symptom of DMD selected from the group consisting of muscle wasting, skeletal deformity, cardiomyopathy, muscle ischemia, cognitive impairment, and impaired respiratory function.

[0187] In some embodiments, administration of an anti-Flt-1 antibody or antigen-binding fragment thereof improves clinical outcomes as measured by the 6-minute walk test, quantitative muscle strength testing, routine exercise capacity testing, Brooke and Vignos limb function scale, pulmonary function tests (forced vital capacity, forced expiratory volume in 1 second, peak expiratory flow rate, peak inspiratory pressure, and peak expiratory pressure), health-related quality of life, knee and elbow flexors, elbow extensors, shoulder abduction, grip strength, sit-up time, North Start Ambulatory assessment, 10-meter walk / run measure, Egen-Classification scale, Gowers score, Hammersmith motor performance, grip strength, range of motion, goniometry, hypercapnia, Nayley Scale of Infant and Young Child Development, and / or caregiver burden scale.

[0188] Combination therapy In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof is administered in combination with one or more additional therapeutic agents. In one embodiment, the additional therapeutic agent is a corticosteroid, such as prednisone. In another embodiment, the additional therapeutic agent is a glucocorticoid, such as deflazacort. In another embodiment, the additional therapeutic agent is follistatin or a recombinant protein thereof. In another embodiment, the additional therapeutic agent is an RNA regulatory therapy. The RNA regulatory therapy can be exon skipping therapy or gene therapy. The RNA regulatory therapy can be, for example, Drispersen, PRO044, PRO045, Eteplirsen (AVI-4658), SRP-4053, SRP-4045, SRP-4050, SRP-4044, SRP-4052, SRP-4055, or SRP-4008. In some embodiments, the additional therapeutic agent is currently used to treat muscular dystrophy. In other embodiments, the additional therapeutic agent can also be used to treat other diseases or disorders. In some embodiments, the known therapeutic agent(s) are administered according to their standard or approved dosing regimen and / or schedule. In some embodiments, the known therapeutic agent(s) are administered according to a regimen that is altered when compared to their standard or approved dosing regimen and / or schedule. In some embodiments, such altered regimens differ from the standard or approved dosing regimen in that one or more unit doses are altered in amount (e.g., decreased or increased) and / or in that administration is altered in frequency (e.g., one or more intervals between unit doses are increased, resulting in less frequency, or decreased, resulting in more frequency). [Example]

[0189] Example 1. Generation and characterization of high affinity anti-Flt-1 antibodies Antibody generation Monoclonal antibodies were generated against soluble Flt-1 using the llama monoclonal antibody method. Briefly, llamas were immunized with recombinant human soluble Flt-1 (purchased from ABCAM) and serum was collected.

[0190] Antibody characterization The antibodies that bound to human and mouse Flt-1 were further characterized for 1) VH family, 2) affinity to Flt-1, 3) IC50, 4) off-rate screening by Biacore assay, 5) cross-reactivity to cynomolgus monkey Flt-1, and 6) binding to VEGF R2 and VEGF R3. Candidate antibodies against human Flt-1 (hFl-1) and mouse Flt-1 (mFlt-1) were characterized as shown in Table 4. [Table 4]

[0191] The pharmacokinetic properties of antibodies 13B4 and 10G12 were tested in mice by intravenous administration of 10 mg / kg of each antibody (Table 5). Data showed that antibody 10G12 was not detectable after 288 hours, while antibody 13B4 was detectable at 672 hours (Figures 1A-1B). [Table 5]

[0192] In vivo efficacy of antibodies Mdx mice (i.e., a mouse model of Duchenne muscular dystrophy) were treated intravenously with either antibody 13B4 or antibody 10G12 at 20 mg / kg twice weekly for one month, starting at 4 weeks of age. Control mice were treated with vehicle alone, an isotype control antibody that does not bind to Flt-1, or a commercially available anti-Flt-1 antibody known as Flt-1:VEGF antagonist (Angio Proteomie, catalog no. AP-MAB0702). Blood was collected 4 days after the fifth intravenous dose to assess serum antibody concentrations at the lowest exposure point. Blood was collected 24 hours after the last dose to assess serum antibody concentrations at the highest exposure point. The highest and lowest concentrations of antibodies 13B4 and 10G12 are shown in Figures 2A and 2B. The concentrations of free antibody 13B4 and free antibody 10G12 in the blood were higher than those of the isotype control antibody and the commercial control antibody at both the highest and lowest exposure points.

[0193] To assess serum free sFlt-1 and VEGF concentrations, blood was collected 24 hours after the fifth intravenous administration and before sacrifice. Administration of antibodies 13B4 and 10G12, as well as a commercial control antibody, significantly reduced serum sFlt-1 concentrations compared with the isotype control antibody (p<0.0001) (Figure 3). Administration of antibodies 13B4 and 10G12 significantly increased serum VEGF concentrations compared with the isotype control antibody (p<0.001) (Figure 4). Administration of the commercial control antibody also significantly increased serum VEGF concentrations compared with the isotype control antibody (p<0.05) (Figure 4).

[0194] Histopathological examination Mice were sacrificed at the end of the 30-day treatment period, and diaphragm and tibialis anterior (TA) muscles were collected and sectioned to determine whether anti-Flt-1 antibodies induced skeletal muscle angiogenesis. Muscle sections were stained for the endothelial cell marker CD31. Significant increases in capillary density were observed in the diaphragms of mice treated with antibodies 13B4, 10G12, or a commercial control antibody compared with those treated with an isotype control antibody (Figures 5A-5D). Data were quantified using automated quantitative imaging software, as shown in Figures 6A-6B. Significant increases in CD31-positive areas were observed in the diaphragms of mice treated with the commercial control antibody (p<0.05), antibody 13B4 (p<0.01), and antibody 10G12 (p<0.0001) compared with those treated with an isotype control antibody. There was also a significant increase in CD31-positive area in the tibialis anterior (TA) muscle of mice treated with antibody 10G12 (p<0.01) compared to the TA muscle of mice treated with an isotype control antibody.

[0195] This experiment demonstrated that administration of Flt-1 antibodies (e.g., 10G12 and 13B4) to mdx mice resulted in a significant increase in endothelial cell proliferation, as well as a decrease in serum soluble Flt-1 and an increase in serum VEGF concentrations. These antibodies demonstrated binding affinities for Flt-1 targets in the pM range (see Table 4), IC50s for Flt-1 binding of less than 100 pM (see Table 4), and greater than 50% rescue of VEGF signaling in bioassays.

[0196] Example 2. Generation and characterization of high affinity anti-Flt-1 antibodies Additional anti-Flt-1 monoclonal antibodies were generated as described above, and these antibodies were further characterized for their binding affinity to the sFlt-1 antigen (by ELISA and Biacore), their competition for VEGF in the sFlt-1:VEGF competition ELISA, and their performance in cell-based assays.

[0197] Antibody characterization - target binding Monoclonal anti-Flt-1 antibodies were analyzed for binding to recombinant sFlt-1 antigen in an ELISA assay (Figure 7). All antibodies showed a dose-dependent increase in binding. The binding affinities of the anti-Flt-1 antibodies to mouse and human Flt-1 antigens were measured using surface plasmon resonance methodology (i.e., Biacore) (Table 6). The antibodies bound to human Flt-1 in the nanomolar range, with antibody 11A11 exhibiting the highest binding affinity for human Flt-1. Biacore analysis also showed that the antibodies did not cross-react with VEGF R2 or VEGF R3 (Table 6), but all antibodies cross-reacted with cynomolgus monkey Flt-1. [Table 6]

[0198] Antibody characterization - competition / antagonism To estimate the potency of the antibodies, the antibodies were analyzed by competitive ELISA using human sFlt-1 and VEGF. The antibody concentrations tested ranged from 0.1 mg / mL to 10,000 ng / mL. A commercially available anti-Flt-1 antibody was used as a control. All antibodies, except for 11A11, were able to block VEGF binding to sFlt-1 (Figure 8).

[0199] Antibody characterization - cell-based assays Human vein endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies 02G07, 11A11, and 13B4. VEGF-induced activation of the cells was examined by determining the phosphorylation status of the VEGF R2 receptor. Data were expressed as % rescue of VEGF R2 receptor phosphorylation relative to phosphorylation of the VEGF R2 receptor in the presence of sFlt-1 alone (e.g., without anti-Flt-1 antibody). The monoclonal antibodies rescued cell activation (i.e., phosphorylation) by antagonizing soluble Flt-1.

[0200] Example 3. Characterization of high affinity anti-Flt-1 antibodies generated by light chain shuffling Light chain shuffling of antibodies 18B6, 11A11 and 13B4, as described in Example 2, was performed to increase the affinity and potency of the candidate antibodies.

[0201] Antibody characterization - target binding The resulting antibodies showed increased affinity for the Flt-1 antigen. For example, the K D The K of antibody 21B3 was increased approximately 10-fold compared to the parent antibody 11A11. D was increased approximately 5-fold over the parent antibody 13B4 (Table 7). [Table 7]

[0202] Antibody characterization - competition / antagonism To estimate antibody potency after light chain shuffling, the antibodies were analyzed by competitive ELISA using human sFlt-1 and VEGF. Antibody concentrations tested ranged from 0.2 mg / mL to 200 ng / mL. The ability of the parent antibody 13B4 to competitively bind to sFlt-1 was compared with that of the antibodies generated by light chain shuffling. All antibodies demonstrated dose-dependent inhibition of VEGF binding to human sFlt-1 by the most effective competitor, clone 21B3 (Figure 10).

[0203] In vivo efficacy To determine the serum half-life and pharmacokinetic properties of the light chain-shuffled antibodies, 125Mice were administered a single 10 mg / kg dose of the light-chain-shuffled antibodies 27H9 and 21B3 and the parental antibody 13B4, labeled with α-glucan. Serum was collected at 0.083, 0.25, 0.5, 1, 4, 8, and 24 hours, and on days 3, 5, 7, 14, 21, and 28 to determine serum antibody concentrations. Serum half-life was decreased for the light-chain-shuffled antibodies compared to the parental antibodies (Figure 11). However, the light-chain-shuffled antibodies exhibited improved pharmacodynamic properties compared to their parental counterparts. For example, antibody 27H9 reached a maximum concentration of 222.4 μg / mL at 0.083 hours, whereas parental antibody 13B4 reached a maximum concentration of 217 μg / mL at 0.5 hours (Table 8). [Table 8]

[0204] Histopathological examination To determine whether the light chain-shuffled and parental antibodies could induce endothelial cell proliferation, mdx mice were treated intravenously with 20 mg / kg of antibody every other week for 4 weeks. Mice were sacrificed at the end of the treatment period, and the diaphragm and tibialis anterior muscles were collected and sectioned to determine whether the antibodies induced skeletal muscle angiogenesis. Muscle sections were stained for the endothelial cell marker CD31. A significant increase in capillary density in the diaphragm was observed in mice treated with antibodies 13B4 and 21B3 compared to the diaphragms of mice treated with an isotype control antibody (Figures 12A-C). Furthermore, there was a significant increase in capillary density in the tibialis anterior muscle of mice treated with the light chain-shuffled antibody 21B3 compared to the tibialis anterior muscle of mice treated with an isotype control antibody (Figures 12D-F).

[0205] The biodistribution of antibodies 27H9, 13B4, and 21B3 in the diaphragm, tibia, and gastrocnemius muscles was 125 This was determined using antibodies labeled with I. The diaphragm showed the highest exposure over time for all antibodies (Figures 13A-C).

[0206] These experiments demonstrated that administration of Flt-1 antibodies (ie, 13B4 and 21B3) to mdx mice resulted in a significant increase in endothelial cell proliferation.

[0207] Example 4. In vivo efficacy of high affinity anti-Flt-1 antibody 21B3 Mdx mice were treated intravenously with 1, 3, 10, or 20 mg / kg of antibody 21B3 or 20 mg / kg of an isotype control antibody twice weekly for one month, starting at 4 weeks of age. Blood was collected four days after the fifth intravenous dose to assess serum antibody concentrations at the lowest exposure point. Blood was collected 24 hours after the last dose to assess serum antibody concentrations at the highest exposure point. The maximum and minimum serum concentrations of antibody 21B3 and the isotype control antibody are shown in Figures 14A and 14B. The maximum and minimum concentrations of antibody 21B3 were dose-dependent and higher than those of the isotype control antibody.

[0208] To assess serum levels of free sFlt-1, blood was collected on days 0, 14, and 28. Administration of antibody 21B3 induced a dose-dependent decrease in serum free sFlt-1 levels. The response was more sustained at doses of 10 and 20 mg / kg, as seen on both days 14 and 28. However, statistically significant decreases were observed in free sFlt-1 levels at doses of 3, 10, and 20 mg / kg compared to free sFlt-1 in mice treated with vehicle alone (FIG. 15).

[0209] To assess serum VEGF levels, blood was collected on days 0, 14, and 28. Administration of antibody 21B3 induced a dose-dependent increase in serum VEGF levels. As observed with free sFlt-1, the response was more sustained at doses of 10 and 20 mg / kg, and on both days 14 and 28. Indeed, a statistically significant increase in serum VEGF was observed at doses of 10 and 20 mg / kg of antibody 21B3 compared to serum VEGF levels in mice treated with vehicle alone ( FIG. 16 ).

[0210] Histopathological examination Mice were sacrificed at the end of the 30-day treatment period, and the diaphragm and tibialis anterior muscles were collected and sectioned to determine whether the anti-Flt-1 antibody induced skeletal muscle angiogenesis. Muscle sections were stained for the endothelial cell marker CD31. A significant increase in capillary density was observed in the diaphragm muscles of mice treated with antibody 21B3 compared to that of mice treated with an isotype control antibody (Figures 17A-17E). Data were quantified using automated quantitative imaging software, as shown in Figure 18. There was a significant increase in the CD31-positive area in the diaphragm muscles of mice treated with 10 mg / kg or 20 mg / kg (p<0.0001) compared to the CD31-positive area in the diaphragms of mice treated with an isotype control antibody.

[0211] A significant increase in capillary density in the tibialis anterior muscle was also seen in mice treated with antibody 21B3 compared to that in mice treated with an isotype control antibody (Figures 19A-19E). Data were quantified using automated quantitative imaging software as shown in Figure 20. There was a significant increase in CD31-positive area in the tibialis anterior muscle of mice treated with 10 mg / kg or 20 mg / kg (p<0.0001) compared to that in the tibialis anterior muscle of mice treated with an isotype control antibody.

[0212] RP-LC / MS characterization The molecular weight of the deglycosylated 21B3 antibody was measured by reverse-phase liquid chromatography / mass spectrometry (RP-LC / MS) (Figure 21A). After the reduction reaction, the molecular weights of the light and heavy chains were measured. The glycosylation pattern of the heavy chain was also measured (Figure 21B).

[0213] These results showed that administration of Flt-1 antibody (ie, 21B3) to mdx mice resulted in a significant increase in endothelial cell proliferation, as well as a decrease in serum soluble Flt-1 and an increase in serum VEGF levels.

[0214] Example 5. Characterization of humanized high affinity anti-Flt-1 antibodies The light-chain-shuffled antibodies described in Example 3 were further modified to introduce sequence mutations into the CDR regions and / or Fc effector regions. These antibodies were evaluated by surface plasmon resonance (e.g., Biacore) to determine their binding properties (Table 9). Antibody 27H9 NG / NA AAA showed approximately two-fold reduced binding affinity for Flt-1. [Table 9-1] [Table 9-2]

[0215] The antibodies were also evaluated in a cell-based assay for their ability to rescue cell activation by antagonizing sFlt-1. Human vein endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies. VEGF-induced activation of the cells was examined by determining the phosphorylation status of the VEGF R2 receptor. The monoclonal antibodies rescued cell activation (i.e., phosphorylation) by antagonizing soluble Flt-1 (Figure 22A), and antibody 27H9 NG / NA AAA (NA+AAA) had comparable potency compared to the unmutated parent antibody (wt).

[0216] Example 6. Antibody optimization Candidate antibodies were analyzed to identify the closest human VH and VL germline sequences, as well as to identify distinct residues in the framework regions and oxidation / isomerization sites within the CDRs. Fab libraries containing human and wild-type residues were constructed and fused to human constant domains. Phage display was applied to identify Fabs with identical or higher dissociation rates (i.e., no loss of affinity) than the parent antibody (21B3). Fabs with the desired dissociation rates were sequenced and compared to human germline sequences, and those with the highest identity (e.g., VH+VL identity >95%) and homology (e.g., homology >96%) were selected for conversion into human monoclonal antibodies. Fabs were also analyzed for unnecessary amino acids. Table 10 provides Fabs ranked by percent human identity, with several clones having up to 97.6% human identity and 98.8% homology. [Table 10]

[0217] Example 7. Characterization of anti-Flt-1 monoclonal antibodies The thermostability of the monoclonal antibodies was analyzed using the Biacore method. At a concentration of 100 μg / mL, each monoclonal antibody was incubated in phosphate-buffered saline at different temperatures for 1 hour. After 1 hour of incubation, the antibody was slowly cooled to 25°C over 2 hours and then incubated at 4°C overnight. The percentage of functional antibody was then measured by determining binding to human Flt-1 using Biacore (see Table 11). The thermostability of the wild-type antibody was consistent with previous experiments. However, except for the 27H6 DG / DA clone, mutations in the VH or VL regions reduced the melting temperature by approximately 2°C. The AAA mutation in the Fc region did not affect the thermostability of the antibody. [Table 11-1] [Table 11-2]

[0218] The binding affinities of the humanized clones were analyzed by Biacore (Table 12). The ability of antibodies 27H4, 27H6, and 27H9 to rescue VEGF signaling in a VEGF:sFlt-1 cell-based assay was measured (Figure 22B). Briefly, human vein endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies 27H4, 27H6, and 27H9. VEGF-induced activation of the cells was examined by determining the phosphorylation status of the VEGF R2 receptor. Data were expressed as % rescue of VEGF R2 receptor phosphorylation relative to the phosphorylation of VEGF R2 receptor in the presence of sFlt-1 alone (e.g., without anti-Flt-1 antibody). The ability of antibodies 27H4, 27H6, and 27H9 to antagonize the binding of VEGF and sFlt-1 (Figure 23) was measured by ELISA. [Table 12]

[0219] Example 8. In vitro testing of anti-Flt-1 antibodies in muscle pathology Mdx mice were treated intravenously with 1, 3, 10, or 10 mg / kg of the anti-Flt-1 antibody 21B3, or 10 mg / kg of an IgG1 isotype control antibody, twice weekly for 6 or 12 weeks, starting at 3 weeks of age.

[0220] To evaluate serum levels of free antibody, blood was collected from mice 4 days after intravenous administration at weeks 2, 4, 7, and 10. Sacrifice samples were collected 24 hours after the final administration (Figure 24). At a dose of 10 mg / kg, there was a statistically significant difference in serum free antibody concentrations compared to the serum levels of mice administered the isotype control antibody at all time points. At a dose of 3 mg / kg, there was a statistically significant difference in serum free antibody concentrations compared to the serum levels of mice administered the isotype control antibody at weeks 4, 7, and 10 and at the time of sacrifice. At a dose of 1 mg / kg, there was a statistically significant difference in serum free antibody concentrations compared to the serum levels of mice administered the isotype control antibody at the time of sacrifice.

[0221] To assess serum concentrations of free sFlt-1, blood was collected from mice 4 days after intravenous administration at weeks 2, 4, 7, and 10. Sacrifice samples were collected 24 hours after the final administration (Figure 25). At a dose of 10 mg / kg, there was a statistically significant difference in serum concentrations of free sFlt-1 compared to the serum concentrations of free sFlt-1 in mice administered an isotype control antibody at all time points. At a dose of 3 mg / kg, there was a statistically significant difference in serum concentrations of free sFlt-1 compared to the serum concentrations of free sFlt-1 in mice administered an isotype control antibody at weeks 4, 7, and 10, as well as at the time of sacrifice. At a dose of 1 mg / kg, there was a statistically significant difference in serum concentrations of free sFlt-1 compared to the serum concentrations of free sFlt-1 in mice administered an isotype control antibody at the time of sacrifice.

[0222] To assess serum VEGF concentrations, blood was collected from mice 4 days after intravenous administration at weeks 2, 4, 7, and 10. Sacrifice samples were collected 24 hours after the final administration (Figure 26). Administration of antibody 21B3 induced a dose-dependent increase in serum VEGF concentrations. At a dose of 10 mg / kg, statistically significant differences in serum VEGF concentrations were observed at all time points compared to mice administered an isotype control antibody. At a dose of 3 mg / kg, statistically significant differences in serum VEGF concentrations were observed at week 7 and at sacrifice. At a dose of 1 mg / kg, statistically significant differences in serum VEGF concentrations were observed at sacrifice compared to mice administered an isotype control antibody.

[0223] Histopathological examination Mice were sacrificed at 6 and 12 weeks of treatment, and the diaphragm, gastrocnemius, and tibialis anterior muscles were collected and sectioned to determine whether treatment with anti-Flt-1 antibody induced angiogenesis and prevented skeletal muscle fibrosis and necrosis.

[0224] angiogenesis Muscle sections were stained for the endothelial cell marker CD31 (Figures 27A-H, 28A-H, and 29A-H). A significant increase in capillary density was seen in all muscle groups tested in mice treated with antibody 21B3 compared to muscles in mice treated with an isotype control antibody. Data were quantified using automated quantitative imaging software. There was a statistically significant increase in CD31-positive area at 6 and 12 weeks in the diaphragm muscles of mice treated with antibody 21B3 at 3 mg / kg (p<0.01) and 10 mg / kg (p<0.0001) compared to the CD31-positive area in the diaphragm muscles of mice treated with an isotype control antibody. There was a statistically significant increase in the CD31-positive area in the gastrocnemius muscle of mice treated with 10 mg / kg of antibody 21B3 (p<0.05) at weeks 6 and 12 compared to the CD31-positive area in the gastrocnemius muscle of mice treated with an isotype control antibody. There was a statistically significant increase in the CD31-positive area in the tibialis anterior muscle of mice treated with 3 mg / kg of antibody 21B3 at weeks 6 (p<0.05) and 12 (p<0.0001) and in mice treated with 10 mg / kg of antibody 21B3 (p<0.0001) at weeks 6 and 12 compared to the CD31-positive area in the gastrocnemius muscle of mice treated with an isotype control antibody (Figures 30A-C).

[0225] fibrosis Muscle sections were further stained for type I collagen by immunohistochemistry (Figures 31A-31H, 32A-32H, and 33A-33H). A significant decrease in type I collagen staining was observed in the diaphragm and gastrocnemius muscles of mice treated with antibody 21B3 compared to those treated with an isotype control antibody. There was a statistically significant decrease in type I collagen staining at week 12 in the diaphragm muscles of mice treated with antibody 21B3 at 1 mg / kg (p<0.0001), 3 mg / kg (p<0.001), and 10 mg / kg (p<0.0001) compared to type I collagen staining in the diaphragm muscles of mice treated with an isotype control antibody. There was a statistically significant decrease in type I collagen staining at week 12 in the gastrocnemius muscle of mice treated with antibody 21B3 at 1 mg / kg (p<0.01), 3 mg / kg (p<0.05), and 10 mg / kg (p<0.001) compared to type I collagen staining in the gastrocnemius muscle of mice treated with an isotype control antibody (Figures 34A-C).

[0226] necrosis The percentage of necrosis present in the gastrocnemius muscle of mice treated with antibody 21B3 was assessed compared to the percentage of necrosis present in the gastrocnemius muscle of mice treated with an isotype control antibody, and a trend toward amelioration of necrosis was observed (Figures 35A-B).

[0227] Example 9. Mapping of epitopes on human sFlt-1 targeted by anti-Flt-1 antibodies 21B3 and 21C6 The peptide-level epitopes of human sFlt-1 targeted by anti-human sFlt-1 monoclonal antibodies (mAbs) 21B3 and 21C6 were determined by hydrogen-deuterium exchange (HDX) mass spectrometry.

[0228] Pepsin digestion and LC-MS For pepsin digestion, 10 μg of sFlt-1, a mixture of sFlt-1 and antibody (21B3) (10 μg:20 μg), or a mixture of sFlt-1 and antibody (21C6) (10 μg:20 μg) was denatured in 0.365 M TCEP and 1.7 M guanidinium chloride (pH 2.5). The mixtures underwent online pepsin digestion, and the resulting peptides were analyzed using a UPLC-MS system consisting of a Waters Acquity UPLC coupled to a MicroTOF-Q2 mass spectrometer (Bruker). Peptides were separated on a 50 mm x 1 mm C8 column with a 19-minute gradient from 5 to 28.5% solvent B (0.1% formic acid in acetonitrile). Solvent A was 0.1% formic acid in water. The solvent mixing valve, injection valve, C8 column, and all connecting stainless steel tubing were immersed in a cooled circulating water bath maintained at 0 °C. Peptide identification was performed by searching MS / MS data against the sFlt-1 sequence in Mascot. Mass tolerances for precursor and product ions were 0.1 Da and 0.2 Da, respectively.

[0229] Deglycosylation 200 μg of human sFlt-1 recombinant protein was incubated with 10 μl of PNGaseF at 37° C. for 4 hours.

[0230] Preparation of Fab Fabs were prepared from two anti-sFlt-1 mAbs (21B3 and 21C6) by papain digestion and protein A capture using the Pierce Fab preparation kit.

[0231] Size Exclusion Chromatography (SEC) To confirm the binding between either native or deglycosylated human sFlt-1 and two anti-human sFlt-1 mAbs (21B3 and 21C6) on SEC, 10 μg of sFlt-1 (either native or deglycosylated) was mixed with 40 μg of anti-sFlt-1 mAb. Native or deglycosylated sFlt-1 alone, anti-sFlt-1 mAb alone, or the complex was injected onto the SEC column with PBS at a flow rate of 0.35 ml / min, while monitoring the migration phase and protein at 280 nm. Binding between Fabs generated from the anti-sFlt-1 mAbs (21B3 and 21C6) and anti-sFlt-1 Fabs and sFlt-1 was also assessed using SEC.

[0232] HDX Ten μL of human sFlt-1 (10 μg), a mixture of sFlt-1 and mAb (21B3) (10 μg:20 μg), or a mixture of sFlt-1 and mAb (21C6) (10 μg:20 μg) was incubated in 90 μL of deuterium oxide-labeled buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4) for 0 s, 30 s, 2 min, 10 min, 1 h, or 4 h. Deuterium exchange was quenched by adding 100 μL of 3.4 M guanidine hydrochloride, 0.73 M TCEP buffer at a final pH of 2.5, followed by pepsin digestion and LC-MS analysis as described above. Mass spectra were recorded in MS-only mode. Raw MS data were processed using HDExaminer software (Sierra Analytics, CA). Deuterium concentrations were calculated using the average mass difference between the deuterated peptide and its native form (t0).

[0233] result To verify that glycan removal did not alter the binding of human sFlt-1 to antibodies, native and deglycosylated sFlt-1 proteins were mixed with anti-human sFlt-1 IgGs (21B3 and 21C6), and complex formation was monitored by size-exclusion chromatography. The data demonstrated that native human sFlt-1 bound completely to the two anti-human sFlt-1 IgGs (21B3 and 21C6), whereas deglycosylated human sFlt-1 bound poorly to anti-human sFlt-1 mAb (21B3) or did not bind to anti-human sFlt-1 mAb (21C6), indicating that deglycosylation disrupts the interaction between human sFlt-1 and antibodies. Therefore, native human sFlt-1 was chosen for HD exchange experiments. Due to heterogeneous glycosylation and the high complexity of the 12 N-linked glycosylation sites, poor sequence coverage was initially achieved in native human sFlt-1. To improve sequence coverage, the glycan masses at each glycosylation site were identified, and high sequence coverage (85.2%) was achieved in native human sFlt-1.

[0234] Native human sFlt-1 was incubated in deuterium oxide alone or in complex with either anti-human sFlt-1 mAb (21B3) or anti-human sFlt-1 mAb (21C6). Deuterium exchange was performed at room temperature for 0, 30, 2, 10, 60, and 240 minutes. The exchange reaction was quenched at low pH, and the protein was digested with pepsin. The deuterium concentrations of identified peptides were monitored by mass shift on LC-MS. Deuterium accumulation curves were plotted for all peptides versus exchange time. While most human sFlt-1 peptides showed identical or similar deuterium concentrations with and without anti-human sFlt-1 mAb (21B3 and 21C6), some peptide moieties had significantly reduced deuterium uptake upon mAb 21B3 or 21C6 binding. Residues 117-129 (corresponding to amino acids 141-153 of SEQ ID NO:90) and 169-182 (corresponding to amino acids 193-206 of SEQ ID NO:90) underwent strong deuterium protection upon binding to anti-human sFlt-1 mAb 21B3, whereas residues 106-114 (corresponding to amino acids 130-138 of SEQ ID NO:90) and 117-124 (corresponding to amino acids 141-148 of SEQ ID NO:90) underwent strong deuterium protection upon binding to anti-human sFlt-1 mAb 21C6. These strongly protected regions were assigned as epitope peptides in the anti-human sFlt-1 mAbs (21B3 and 21C6) and are highlighted in blue in the differential heat maps shown in Figures 36 and 37. MS / MS spectra for identified peptides containing amino acid residues from the epitope regions are shown in Figures 38A-E. Peptides 115 to 124 correspond to amino acids 139 to 148 of SEQ ID NO:90, peptides 115 to 129 correspond to amino acids 139 to 153 of SEQ ID NO:90, peptides 154 to 182 correspond to amino acids 178 to 206 of SEQ ID NO:90, peptides 175 to 180 correspond to amino acids 119 to 204 of SEQ ID NO:90, and peptides 104 to 114 correspond to amino acids 128 to 138 of SEQ ID NO:90.

[0235] conclusion A sequence coverage of 85.2% was obtained for human sFlt-1. Residues 117-129 and 169-180 underwent strong deuterium protection upon binding with anti-human sFlt-1 mAb 21B3, whereas residues 106-114 and 117-124 underwent strong deuterium protection upon binding with anti-human sFlt-1 mAb 21C6. These strongly protected regions were assigned as epitope peptides in the corresponding anti-human sFlt-1 mAbs.

[0236] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is not intended that the scope of the present invention be limited to the above detailed description of the invention, but rather is set forth in the claims that follow. [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 14]

Claims

1. A polynucleotide encoding an anti-Flt-1 antibody or antigen-binding fragment thereof, (a) a variable light chain (VL) CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:32, a variable heavy chain (VH) CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:76, and a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:71; (b) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:27, a variable heavy chain (VH) CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:85, and a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:72; (c) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, a variable heavy chain (VH) CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:84, and a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:71; (d) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:28, a variable heavy chain (VH) CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:8, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17; a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:86, and a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:73; or (e) a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, a variable heavy chain (VH) CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:6, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:16; a light chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:83, and a heavy chain comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

70. A polynucleotide comprising:

2. An expression vector comprising the polynucleotide of claim 1.

3. An isolated cell comprising the polynucleotide of claim 1.

4. An isolated cell comprising the expression vector of claim 2.

5. A method for producing an anti-Flt-1 antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, comprising culturing the isolated cell of claim 4.

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